[Objective] Inositol, also named cyclohexanol, is a kind of bioactive sugar alcohol. It was widely used in pharmaceutical, food and feed industries. To obtain the microbial cell factory that produced more inositol, the recombinant Saccharomyces cerevisiae was constructed by metabolic engineering. [Methods] Positive and negative regulation of inositol biosynthesis was modified. The Inositol-3-phosphate synthase gene (ino1) was overexpressed in S. cerevisiae. Meanwhile, the transcription inhibitor gene (opi1) that inhibits the biosynthesis of inositol and the antibiotic resistance gene (kanMX) for screening strains were knocked out of recombinant S. cerevisiae. Inositol content in fermentation broth of engineered strains was detected by Gas chromatography. [Results] Biological safety engineered strain S. cerevisiae was constructed and shake flask fermentation results showed the yield of inositol was 1.021 g/L. [Conclusion] Over-expression of ino1 gene and opi1 gene knocked out could efficiently enhance the productivity of inositol in S. cerevisiae. The results provided the foundation for industrial application of inositol production by microbial fermentation.