This study was focused on the development of oil-producing microorganisms to generate lipids. The yeast Rhodotorula mucilaginosa (R. mucilaginosa) was selected for liquid-state cultivation, and the conditions for growth of the yeast cells were assessed. Additionally, the relationships between different nutrient elements and the growth of R. mucilaginosa were explored. The lipid accumulation of R. mucilaginosa is increased under nitrogen-restricted conditions. As the concentration of the carbon source increases, the accumulation of lipids is increased. However, if the carbon source concentration is further increased, the growth of yeast is inhibited. From a large-scale liquid fermentation culture with a fixed inoculation amount of 5%, and from a batch of culture experiments, it was determined that a suitable oil-producing culture was obtained on the 6th day, and the optimum conditions involved a carbon source concentration of 60 g/L, a nitrogen source concentration of 0.5 g/L, and a KH2PO4 concentration of 7.0 g/L. After utilizing different carbon sources in this study, it was found that glucose was the carbon source most conducive to the accumulation of R. mucilaginosa lipids. In addition, the extraction method and solvent for the extraction of R. mucilaginosa lipids were chosen. The acid-heat method using the green organic solvent ethyl acetate exhibited the best performance for extraction of yeast lipids under environmentally friendly and safe conditions. The analysis of lipids showed that the fatty acids obtained primarily contained C16:0, C18:1 and C18:2, and especially C18:1 (41%) lipids, indicating that R. mucilaginosa lipids are a good bio-oil source for the production of biodiesel.