The invention discloses a culture medium for improving the hydrogen production amount of Chlamydomonas reinhardtii, and its preparation method. The culture medium provided by the invention is composed of NH4Cl, MgCl2.6H2O, K2HPO4, KH2PO4, a Tris base, H3BO4, ZnCl2, MnCl2.4H2O, CoCl2.6H2O, CuCl2.2H2O, (NH4)6Mo7O24.4H2O, FeCl2.4H2O and glacial acetic acid. Experiments in the invention prove that the hydrogen production amount of the Chlamydomonas reinhardtii in the sulfur-lack and calcium-lack TAP culture solution is about 2 / 3 higher than the hydrogen production amount of the Chlamydomonas reinhardtii in present sulfur-lack systems after optimizing the formulas of present hydrogen desorption culture mediums (sulfur-lack TAP culture solutions), and a culture medium component is omitted, so the cost is reduced.