Sandalwood oil is a valuable resource derived from Santalum album. It has antibacterial, cosmetic, and sedative effects. -Santalene is the precursor of -santalol, the main component of sandalwood oil. Yarrowia lipolytica is an oleaginous yeast, which has been metabolically engineered to produce valuable compounds such as terpenoids and biofuel. This study presents a method for the heterologous synthesis of -santalene by Y. lipolytica. Using Y. lipolytica ATCC 201249, a codon-optimized plant-origin -santalene synthase (STS) was integrated into the genome, and a yield of 5.19mg/L -santalene was obtained after fermentation. Upstream genes in the MVA pathway (ERG8, ERG10, ERG12, ERG13, ERG19, ERG20, HMG1, and tHMG1) were overexpressed, and we found that the key genes ERG8, HMG1, and tHMG1 can increase the supply of FPP and the yield of -santalene. ERG8 and HMG1 were overexpressed in multiple-copy formats, and the plasmid pERG8HMG1 and ERG8-HMG1 expression modules were optimized as single-copy and multiple-copy formats, respectively. The overexpression of single-copy plasmid pERG8HMG1 led to -santalene yield of 13.31mg/L. The optimal feeding strategy was determined by initial carbon source concentration optimizations and five feeding methods. Using 50g/L glucose as the initial carbon source, maintaining the carbon source concentration at 5-20g/L during the feeding process is most conducive to increased production. These results were verified in a 5-L fermenter by batch and fed-batch fermentation. The OD of fed-batch fermentation broth reached 79.09, and the production of -santalene reached 27.92mg/L; 5.38 times of the initial yield, without by-products farnesol and trans--bergamotene.