Squalene has antioxidant, anti-cancer, and anti-radiation properties and is widely used in the cosmetic and pharmaceutical industries. In this study, squalene synthesis in Candida tropicalis was enhanced via combinatorial metabolic engineering strategies to rebuild pathways. By overexpressing the natural squalene synthase gene CtERG9 under the strong promoter PGAP1 in C. tropicalis DC03, squalene titer was increased by 12.1%. Replacing the natural gene CtERG9 in C. tropicalis with the squalene synthase gene ScERG9 from Saccharomyces cerevisiae increased squalene titer by 47.3%. The squalene titer in the engineered C. tropicalis CS13-D was increased by 15.4% by fusing the genes ScERG9 and ERG20. Deletion of one copy of the diacylglycerol diphosphate phosphatase gene DPP1, the geranylgeranyl pyrophosphate synthase gene BTS1, and the squalene epoxidase gene ERG1 in C. tropicalis CS19E increased squalene titer by 1.36-fold compared to control. Down-regulation of the transcription level of the single-copy gene ERG1 in C. tropicalis CS19E-G using the weak promoter PGPM1 resulted in a 38.5% increase in squalene titer. The squalene titer of C. tropicalis CS19E-G reached 433.06 mg/L in a 30-L fermenter, which was 13.5-fold higher than that of C. tropicalis DC03. This combinatorial strategy has the potential for efficient production of other high-value chemicals in C. tropicalis and other yeasts.