The valorization of converting defatted silkworm pupae (DSWP) into microbial lipids by the oleaginous yeast Yarrowia lipolytica was investigated. To obtain the best hydrolysis of DSWP and obtain the most final production after cultivation, the method of ultrasound-assisted enzymatic pretreatment in the presence of 1-butyl-3-methylimidazolium chloride salt ([Bmim] Cl) followed with trypsin hydrolysis was applied to pretreat the DSWP. Under the optimal conditions by response surface methodology, the highest weight loss achieved 84.97%, the hydrolysis degree (32.35%) and lipid yield (3.71 g/L) have increased by 6.0- and 1.3-fold, respectively. When adding amount of defatted silkworm pupae hydrolysate (DSWPH) was 4 g/L, the yeast could produce lipid efficiently. Besides, the cost is much lower than the peptone. The replacement of peptone with DSWP for lipid production by Y. lipolytica W29 efficiently cut down the cost of microbial lipid production. The lipid concentration of yeast grew on medium with 4 g/L DSWPH adding amount has increased by threefold compared with control group, while the citric acid concentration was decrease to 0.27-fold. Given the large amount of fatty acids accumulation, instead of citric acid accumulation, the DSWPH could replace the industrial nitrogen source in the future. Practical applications: The developed enzymatic pretreatment method has changed the structural of DSWP assisted by using ultrasound and [Bmim] Cl, which application is suitable for the parallel utilization of the nutrient-rich biomass and solving problems of the environmental corruption which caused by the DSWP. It covers more analytes and is more effective than other previously reported methods. Especially an effective, cheap, and safe degradation of DSWP into small moleculars is highly desirable. As a suitable nitrogen source replacement of peptone, the DSWPH is better for lipid production. The novel culture medium was good to the lipid production of Y. lipolytica W29 and has reduced the cost of microbial lipid.