De Forest's father, president of Talladega College in Alabama, had hoped that his son would also become a minister, but early in life Lee had shown an intense interest in inventing. As preparation for what his father hoped would be a religious career, de Forest was enrolled at Mount Hermon school in Massachusetts. The school’s emphasis on hard work (students did most of the chores in order to defray the expenses) left de Forest little time for inventing, and he was unhappy there. Nevertheless, after graduation he was able to convince his parents to allow him to attend the Sheffield Scientific School at Yale University. Eventually, he graduated from Yale University in 1896, obtaining his Ph. D. degree there three years later.
De Forest's first position was in the experimental telephone laboratory of the Western Electric Company in Chicago, and in 1900 he began active work in wireless telegraphy for the Armour Institute of Technology there. In 1902 he founded the American De Forest Wireless Telegraph Company, Jersey City, New Jersey, which did a thriving business in providing wireless telegraphy apparatus for ocean vessels and the United States government; it was succeeded in 1906 by the United Wireless Telegraph Company.
De Forest was chief research engineer for the Federal Telegraph Company in San Francisco from 1911 to 1912. In the latter year he sold the rights to the Audion amplifier as a telephone repeater to the American Telegraph and Telephone Company, thus making possible transcontinental telephony.
In 1915 De Forest transmitted a voice from Arlington, Virginia, to a receiving station atop the Eiffel Tower in Paris, and in 1916 he inaugurated news broadcasts by radio.
De Forest next turned to the development of talking motion pictures. By 1923 he had perfected a method for sound-on-film pictures, called the phonofilm, which he demonstrated for the first time at the Rivoli Theatre in New York. Later he had a part in the development of television and high-speed facsimile transmission.
In 1934 he founded the Lee De Forest Laboratories for research and the manufacture of radiotherapy apparatus.
His autobiography, Father of Radio, appeared in 1950.
Although raised in a strongly religious Protestant household, de Forest later became an agnostic.
De Forest was a conservative Republican and fervent anti-communist and anti-fascist.
De Forest married Lucile Sheardown in 1907, they divorced in 1907. Then he married Nora Stanton Blatch, a civil engineer, they divorced in 1911. After that he married Mary Mayo, a singer on December, 1912, they divorced in 1929. Finally, he married Marie Mosquini, an actress, in 1930. He had a child from the second marriage - Harriot Stanton de Forest, he had children from the third marriage - two daughters and one son.