Born near Americus, Georgia, he was still an infant when his family moved to Florida.

Fletcher studied law at Vanderbilt University in Nashville, Tennessee and after graduating in 1880 he was admitted to the bar the following year and set up a law practice in the city of Jacksonville, Florida. Fletcher became active in municipal politics and was elected to city council in 1887 and served as mayor from 1893 to 1895. In 1893 he was elected to the State house of representatives.

In 1909, Duncan U. Fletcher was elected to the United States Senate as a member of the Democratic Party where he served until his death in 1936. He served on a number of government committees including the high profile Chairmanship of the United States Senate Senate Banking and Currency Committee in 1932 with a mandate to examine the causes of the Wall Street Crash of 1929. His committee, generally known as the Pecora Commission, began a major process of reform of the American financial system and resulted in the passage of the Securities Act of 1933 and the Securities Exchange Act of 1934 that instituted disclosure laws for corporations seeking public financing plus the 1935 formation of the U.S. Securities and Exchange Commission as a mechanism to enforce the provisions of the new Acts.

Senator Fletcher died in Washington, D.C. and was interred in the Evergreen Cemetery in Jacksonville, Florida. The Duncan U. Fletcher High School in Neptune Beach, Florida and the Duncan U. Fletcher Middle School in Jacksonville Beach were named in his memory.