Objectives: To develop the biosynthetic methods of 2-furfurylthiol， this study aimed to achieve the heterologous expression and purification of the key C-S lyase Cys3 responsible for 2-furfurylthiol biosynthesis in Escherichia coli and to build and optimize the optimal conditions of 2-furfurylthiol biosynthesis by catalysis of Cys3. Methods: The C-S lyase Cys3 was cloned from Saccharomyces cerevisiae isolated from the sesame-flavor Baijiu starter culture and was over-expressed in E. coli. The recombinant protein of Cys3 was purified using nickel column affinity chromatography， and the elution condition were optimized for purified protein preparation. The enzymatic catalysis method for 2-furfurylthiol biosynthesis was built using the recombinant Cys3 and furfural and L-cysteine as substrates. The enzymatic reaction parameters were optimized by the single-factor experiment and response surface methodology to determine the optimal reaction conditions. Results: The recombinant Cys3 protein was successfully expressed in E. coli. 200 mmol/L imidazole buffer was determined as the optimal elution condition， by which the recombinant protein of Cys3 (42 ku， 6.17 mg/mL) was purified from E. coli. After optimization， the optimal catalytic conditions for 2-furfurylthiol biosynthesis were determined as follows: 188 μg/mL Cys3， pH 6.7， 16.5 mmol/L furfural， 16.5 mmol/L L-cysteine， 81.9 μmol/L pyridoxal phosphate， and reaction at 50 ℃ for 16.4 h. The maximum yield of 2-furfurylthiol reached 133.8 μmol/L under the optimal conditions. Conclusions: This study achieved the heterologous expression and purification of the key C-S lyase Cys3 for 2-furfurylthiol synthesis and established the optimal parameters for its catalysis in 2-furfurylthiol biosynthesis. The results provide an important technical support for the enzymatic biomanufacturing of 2-furfurylthiol.