Biodiesel is a renewable, easily biodegradable energy. However, due to the rising cost of its raw materials such as vegetable oils, using of biodiesel is restricted. Microbial lipid has many advantages like short production cycle, high production efficiency, and low cost, can be used to provide cheap and ample raw materials for biodiesel production. In this study, Rhodotorula glutinis was treated with ultraviolet radiation to get mutants and then screened by spectrophotometry. RG-UV02 was identified as a high yield oleaginous strain and exhibited a good genetic stability. Optimal fermentation conditions were obtained as follows: 20% dissolved oxygen, 10% inoculum size, 100 g/L glucose and C/N ratio was 154. Under such conditions, dry biomass, lipid concentration and lipid content of RG-UV02 reached 11.583 g/L, 3.535 g/L and 30.51%，respectively, which raised by 10.77%, 36.86% and 23.52% than these before optimizing. Gas chromatography-mass spectroscopy analysis showed that the compositions of RG-UV02 lipid were similar to these of vegetable oils. Chemical modification improved the affinity of the porcine pancreatic lipase with the substrate. The enzyme activity recovery rate of immobilized chemical modification porcine pancreatic lipase was 47.90%. The calculation result showed that immobilized chemical modification porcine pancreatic lipase catalyzed 1g RG-UV02 lipid to synthesize 183.8 mg biodiesel, the yield rate of transesterification was 35.55%.