Currently, the biosynthesis of nanomaterials by organisms is attracting considerable attention because of the sustainable and environmentally friendly nature of the reactions involved in this process compared with those in the conventional nanomaterial synthesis. However, the manipulation and control of nanomaterial biosynthesis remain difficult because of the lack of knowledge about the biosynthetic mechanisms. In the present study, we elucidated the selenium (Se)-precursor and Se metabolic flux in the biosynthesis of cadmium-selenium quantum dots (CdSe QDs) in Saccharomyces cerevisiae and improved the cells' ability to biosynthesize CdSe QDs through gene modification based on the regulation mechanism. By deleting the genes involved in Se metabolism and measuring seleno-amino acids, we identified selenocysteine (SeCys) as the primary Se-precursor in the intracellular biosynthesis of CdSe QDs. Further studies demonstrated that the selenomethionine (SeMet)-to-SeCys pathway regulates CdSe QD biosynthesis. Knowledge of the regulatory pathway allowed us to enhance SeMet synthesis by overexpression of the MET6 gene, and an increased CdSe QD yield was realized in the engineered cells. Understanding the mechanism of CdSe QD biosynthesis helped to determine the relationship between nanocrystal formation and biological processes, and offers a new perspective to manipulation of nanomaterial biosynthesis.