Although Barnard was born into a poor family in Nashville, Tennessee, and received little formal education, he developed a great interest in astronomy and also became familiar with photographic techniques from his work in a portrait studio. He managed both to study and instruct at Vanderbilt University from 1883 to 1887. From 1888 he worked at the Lick Observatory until in 1895 he became professor of astronomy at Chicago and was thus able to work at the newly established Yerkes Observatory.

Barnard was a keen observer and had detected more than ten comets by 1887 and several more in subsequent years. In 1892 he became the first astronomer after Galileo to discover a new satellite of Jupiter, subsequently named Amalthea, which lay inside the orbits of the four Galilean satellites and was much smaller and fainter. In 1916 he discovered a nearby red star with a very pronounced proper motion of 10.3 seconds of arc per year: in 180 years it will appear to us to have moved a very considerable distance, equal to the diameter of the Moon. The star is now called Barnard's star.

Barnard's other discoveries included various novae, variable stars, and binary stars. He was also one of the first to appreciate that dark nebulae were not areas of the sky containing no stars at all (as William Herschel had thought) but, as Barnard and Max Wolf demonstrated, were enormous clouds of dust and gas that shielded the stars behind them from our view. By 1919 he had discovered nearly 200 such nebulae.