Diosgenin (DSG) is a naturally occurring steroidal saponin with a variety of biological activities that is also an important precursor for the synthesis of various steroidal drugs. The traditional industrial production of DSG is based on natural plant extraction and chemical processing. However, the whole process is time-consuming, laborious, and accompanied by severe environmental pollution. Therefore, it is necessary to develop a more convenient and environmentally-friendly process to realize the green production of DSG. In our previous work, we achieved de novo synthesis of DSG in Saccharomyces cerevisiae using glucose as the carbon source. However, DSG production was only at the milligram level, which is too low for industrial production. In this work, we further developed yeast strains for DSG overproduction by optimizing the synthesis pathway, fine-tuning pathway gene expression, and eliminating competing pathways. Cholesterol 22-hydroxylase was used to construct the DSG biosynthesis pathway. The optimal ratio of cytochrome P450 (CYP) to cytochrome P450 reductase (CPR) associated with DSG synthesis was screened to increase DSG production. Weakening the expression of the ERG6 gene further increased DSG synthesis and reduced the formation of by-products. In addition, we investigated the impact of DSG accumulation on yeast cell physiology and growth by transcriptome analysis and found that the multidrug transporter PDR5 and the sterol-binding protein PRY1 contributed to DSG production. Finally, we obtained a DSG titer of 2.03 g/L after 288 h of high-cell-density fed-batch fermentation using the engineered strain LP118, which represents the highest DSG titer reported to date for a yeast de novo synthesis system.