Although Saitozyma podzolica Zwy2-3 can use the enzymatic hydrolysate of corn stalks treated with an ammonium carbonate-steam explosion (EHCS-ACSE) as a substrate for lipid accumulation, the inefficient conversion of sugars from EHCS-ACSE into lipids necessitates the further optimization of fermentation parameters. Response surface design was used to optimize the primary fermentation parameters. Under the optimized conditions of the reducing sugar concentration of 89.44 g/L, yeast extract concentration of 3.88 g/L, rotational speed of 219 rpm, and incubation time of 122 h, the maximum lipid production achieved 11.45 g/L, which was 2.28 times higher than the results of the previous study. In addition, lipid profiling showed the presence of four fatty acid methyl esters, with the highest percentage being 61.84% oleic acid, followed by 21.53% palmitic acid, 13.05% stearic acid, and 3.58% linoleic acid. It is noteworthy that the composition and relative abundance of microbial lipids remained constant under different culture conditions. The characteristics of Zwy2-3 biodiesel, such as the iodine value (62.09), cetane number (59.29), density (0.87 g/cm3), and oxidation stability (35.53), meet the international standards (ASTM D6751-02 and EN 14214) for biodiesel. The present study further demonstrated that S. podzolica Zwy2-3 can efficiently utilize EHCS-ACSE for microbial lipid accumulation, and its lipids have favorable qualities that make them suitable for biodiesel production.