Flagler became a partner in the newly organized D. M. Harkness and Company with his half-brother, Dan Harkness in 1852. The following year, on November 9, he married Mary Harkness. On March 18, 1855, their first child, Jennie Louise, was born. Jennie Louise lived until 1889, when at the age of 34, she died following complications from child birth. A second child, Carrie, was born on June 18, 1858. She died three years later. On December 2, 1870, the Flaglers' only son, Harry Harkness Flagler, was born.

Flagler founded the Flagler and York Salt Company, a salt mining and production business in Saginaw, Michigan in 1862 with his brother-in-law Barney York. By 1865, the end of the Civil War caused a drop in the demand for salt and the Flagler and York Salt Company collapsed. Heavily in debt, Flagler returned to Bellevue, Ohio. He had lost his initial $50,000 investment and an additional $50,000 he had borrowed from his father-in-law and Dan Harkness.

The next year Flagler reentered the grain business as a commission merchant. Flagler had become acquainted with John D. Rockefeller, who worked as a commission agent with Hewitt and Tuttle for the Harkness Grain Company. By the mid 1860s, Cleveland had become the center of the oil refining industry in America and Rockefeller left the grain business to start his own oil refinery. In 1867, Rockefeller, needing capital for his new venture, approached Flagler. Flagler obtained $100,000 from a relative on the condition that Flagler be made a partner. A Rockefeller, Andrews and Flagler partnership was formed with Flagler in control of Harkness' interest. On January 10, 1870, the Rockefeller, Andrews and Flagler partnership emerged as a joint-stock corporation named Standard Oil and by 1872, Standard Oil led the American oil refining industry, producing 10,000 barrels per day. Five years later Standard Oil moved its headquarters to New York City, and the Flaglers moved to their new home at 509 Fifth Avenue in New York City.

By 1878, Flagler's wife, who had always struggled with health problems, was very ill. On advice from Mary's physician, she and Flagler visited Jacksonville, Florida for the winter. Mary's illness grew worse, however, and she died on May 18, 1881 at age 47. Two years after Mary's death, Flagler married Ida Alice Shourds. Soon after their wedding, the couple traveled to St. Augustine, Florida where they found the city charming, but the hotel facilities and transportation systems inadequate. Flagler recognized Florida's potential to attract out-of-state visitors. Though Flagler remained on the Board of Directors of Standard Oil, he gave up his day-to-day involvement in the corporation in order to pursue his interests in Florida. He returned to St. Augustine in 1885 and began construction on the 540-room Hotel Ponce de Leon. Realizing the need for a sound transportation system to support his hotel ventures, Flagler purchased the Jacksonville, St. Augustine & Halifax Railroad, the first railroad in what would eventually become the Florida East Coast Railway.

Flagler originally intended for West Palm Beach to be the terminus of his railroad system, but during 1894 and 1895, severe freezes hit the area, causing Flagler to rethink this original decision. Sixty miles south, the town today known as Miami was reportedly unharmed by the freeze. To further convince Flagler to continue the railroad to Miami, he was offered land from private landowners, the Florida East Coast Canal and Transportation Company, and the Boston and Florida Atlantic Coast Land Company, in exchange for laying rail tracks.

Flagler's railroad, renamed the Florida East Coast Railway in 1895, reached Biscayne Bay by 1896. Flagler dredged a channel, built streets, instituted the first water and power systems, and financed the town's first newspaper, the Metropolis. When the town incorporated in 1896, its citizens wanted to honor the man responsible for its growth by naming it "Flagler." He declined the honor, persuading them to use an old Indian name, "Miami". In 1897, Flagler opened the exclusive Royal Palm Hotel in Miami.

Flagler's second wife, Ida Alice, had been institutionalized for mental illness since 1895. In 1901, the Florida Legislature passed a bill that made incurable insanity grounds for divorce, opening the way for Flagler to remarry. On August 24, 1901, Flagler married Mary Lily Kenan and the couple soon moved into their Palm Beach estate, Whitehall. Built as a wedding present to Mary Lily in 1902, Whitehall established the Palm Beach season for the wealthy of America's Gilded Age.

By 1905, Flagler decided that his Florida East Coast Railway should extend from Biscayne Bay to Key West, a point 128 miles past the end of the Florida peninsula. At the time, Key West was Florida's most populated city and Flagler wanted to take advantage of additional trade with Cuba and Latin America as well as the increased trade with the west that the upcoming Panama Canal would bring. In 1912, the Florida Overseas Railroad was completed to Key West. In 1913, Flagler fell down a flight of stairs at Whitehall. He never recovered from the fall and died of his injuries on May 20 at 84 years of age. He was buried in St. Augustine alongside his daughter, Jennie Louise and first wife, Mary Harkness.