Abstract: In this study, crude glycerol, a by-product in the process of biodiesel preparation from waste cooking oil, was used as carbon source to produce microbial lipid by Rhodosporidium toruloides. Through the optimization, the optimized fermentation conditions were determined as follows: seed resuscitation time of 24 h, enrichment time of 10 h, inoculation rate of 6%, initial crude glycerin concentration of 60 g/L, ratio of carbon to nitrogen of 60, organic nitrogen ratio to total nitrogen of 75%, fermentation volume of 100 mL, fermentation temperature of 30 ℃ and fermentation time of 6 d. Under the optimal condition, lipid production and lipid yield rate reached 10.56 g/L and 0.229, respectively. Compared with inorganic ammonium sulfate, the addition of organic peptone significantly increased the lipid content and lipid production. The performance of biodiesel was estimated by measuring the fatty acid composition of lipid and using Hoekman equation, which met all of the standards of China, the United States and the European Union. This study can not only provide a new strategy for the reutilization of crude glycerol, but also provide a economical raw material for the production of biodiesel.