Retinol, the main active form of vitamin A, plays a role in maintaining vision, immune function, growth, and development. It also inhibits tumor growth and alleviates anemia. Here, we developed aSaccharomyces cerevisiaestrain capable of high retinol production. Firstly, the de novo synthesis pathway of retinol was constructed inS. cerevisiaeto realize the production of retinol. Second, through modular optimization of the metabolic network of retinol, the retinol titer was increased from 3.6 to 153.6 mg/L. Then, we used transporter engineering to regulate and promote the accumulation of the intracellular precursor retinal to improve retinol production. Subsequently, we screened and semi-rationally designed the key enzyme retinol dehydrogenase to further increase the retinol titer to 387.4 mg/L. Lastly, we performed two-phase extraction fermentation using olive oil to obtain a final shaking flask retinol titer of 1.2 g/L, the highest titer reported at the shake flask level. This study laid the foundation for the industrial production of retinol.