His parents emigrated from Switzerland to the United States, where Nef studied chemistry at Harvard University until 1884. Upon graduation, he joined Adolf von Baeyer at the University of Munich, where he received his Ph.D. in 1887.
He was a professor at Purdue University from 1887 till 1889 and at Clark University from 1889 till 1892. In 1892 Nef joined the newly formed University of Chicago as professor of chemistry, where he spent the rest of his academic career.
His son John Ulric Nef (1899–1988) became a professor of economic history and published several books.