The exact site of the library during its earliest years is not known, but the university from its foundation in 1477, was located on what became known as "Student Island" in the Fyris River, where the academy mill – now the provincial museum – was later built.

In 1566, King Eric XIV donated the old chapter house, south of the Uppsala Cathedral, to be used for lectures.

The most famous example is the Codex Argenteus (the "Silver Bible"), most of what remains of Bishop Ulfilas's translation of the New Testament into Gothic, which was looted in Prague.

Another example is the Copernicana, the main part of the library of astronomer Nicolaus Copernicus, looted by the Swedish Army in Kraków.

The so-called Emperor's Bible was lost when Swedish troops occupied Goslar in Germany, and eventually also ended up in Uppsala University Library.

It is one of the largest libraries of books concerned with the history of science and medicine, and a manuscript collection mostly of letters from notable scientists.