The traditional synthesis method of (S)-nicotine suffers from complex steps and relies on multiple chemical catalysts. In order to realize the green synthesis of (S)-nicotine, a chemoenzymatic cascade pathway was designed in this study. Firstly, a coupling system of imine reductase (IRED) and formate dehydrogenase (FDH) was constructed to synthesize (S)-nornicotine using myosmine as the substrate; then (S)-nicotine was prepared via the Eschweiler-Clarke reaction of (S)-nornicotine. The enzyme-containing cells and enzyme solution were obtained via the expression of IRED and FDH in E. coli hosts and the effects of pH and temperature on the enzyme activity of IRED and FDH were investigated. The reaction conditions for the enzymatic synthesis of (S)-nornicotine were optimized, and the yield of (S)-nornicotine was>98% with enantiomeric excess (e.e.) value of >99% at 30 ℃ and pH 7.5 with the addition of myosmine at a mass concentration of 30 g/L, 600 U/L IRED-containing cells, 900 U/L FDH-containing cells, 0.6 mmol/L NADP+ and sodium formate at a mass concentration of 90 g/L. Finally, (S)-nicotine was obtained by chemical methylation of (S)-nornicotine, with a yield and e.e. value of above 99%.