Abstract: This experiment investigated the ability of an amino peptidase gene dmpA from microcycomobasidiomycete MCA4210 (Cystobasidium minutum MCA4210) to catalyze the synthesis of L-carnosine, and constructed a genetically engineered strain to catalyze the synthesis of L-carnosine with L-histidine and β-alanine methyl ester hydrochloride as substrate, aiming to improve the synthesis efficiency of L-carnosine. Codon-optimized DmpA was expressed in E. coli BL21 (DE3) and add a maltose binding protein (MBP) tag further improved solubility and enzyme activity, yielding BL21 (DE3)-MBP-DmpA. Then the recombinant strain BL21(DE3)-MBP-DmpA was further investigated to optimize induced expression and transformation conditions. The results indicated that the target gene fragment was successfully fused with MBP, yielding the recombinant strain BL21(DE3)-MBP-DmpA. After optimization of the induction conditions, the crude enzyme activity reached 145 U. Through optimization of the transformation conditions, the L-carnosine production reached as high as 55.72 g/L, with a yield of 54.54%. This study successfully expressed DmpA from Cystobasidium minutum MCA 4210 in E. coli, demonstrating its L-carnosine synthesis capability. The MBP tag and condition optimizations efficiently improved production, achieving the highest L-carnosine yield reported to date. These findings provided a strong foundation for industrial-scale L-carnosine production using genetically engineered bacteria and highlighted promising application potential.