In this study, the feasibility of banana straw (BS) hydrolysate as carbon source and reutilizing the pretreated liquor (PL) of BS in the Rhodosporidium toruloides fermentation was explored for the first time. When BS hydrolysate was used as the carbon source, total biomass concentration, lipid concentration, and lipid content under optimal conditions reached 15.52 g/L, 5.83 g/L, and 37.56% (w/w), respectively, which was similar to the results of pure sugar control. After detoxification, 50% PL can be returned to enzymatic hydrolysis and fermentation, and total biomass concentration, lipid concentration, and lipid content can reach 15.14 g/L, 5.59 g/L, and 36.91% (w/w). Then, ethanol stillage (ES) was used as the nitrogen source. The NaCl and glycerol of ES could promote lipid accumulation, reaching 7.52 g/L under optimized conditions. Finally, microbial lipid production from BS hydrolysate and ES without any additional nutrients was investigated, and the maximum total biomass concentration, lipid concentration, and lipid content were 13.55 g/L, 4.88 g/L, and 36.01% (w/w), respectively. Besides, the main compositions of microbial lipid produced were C16 and C18, and the biodiesel production from the microbial lipid could meet Chinese and US standard through theoretical numerical calculation.