Sterols are crucial functional components for eukaryotic cell membrane. Due to versatile activities, sterols show wide applications in food and pharmaceutical industries. Ergosterol not only reflects cell growth but also serves as the precursor for manufacturing steroid drugs. To date, the ergosterol biosynthetic pathway in yeast has been reported, and the industrial production of ergosterol is achieved by yeast fermentation or extraction from fungal mycelia. Here, we summarize its biosynthesis, regulation, transportation, and subcellular location of enzymes in yeast. In particular, we review the regulation of ergosterol biosynthesis at transcriptional, translational and post-translational levels. Furthermore, we advocate metabolic engineering and fermentation strategies for high-level production of ergosterol. This study may provide evaluable insights into metabolic engineering of yeast for scaled-up fermentation production of ergosterol or beyond.