To investigate the expression and immobilization of recombinant cis-epoxysuccinate hydrolase (ESH), and its application in the biological production of l-(+)-tartaric acid. E. coli BL21 (DE3)/pET11a-ESH (His) was engineered to express recombinant ESH. The enzyme had an activity of 262 U mg(-1). The recombinant ESH was immobilized on agarose Ni-IDA matrix with metal ion affinity interaction to improve its thermostability and pH stability. The immobilization efficiency and activity yield were 94 and 95%, respectively. The specific catalytic efficiency of immobilized ESH was 104 mg U-1 h(-1) during the continuous enzymatic production process. ESH with a histidine tag was immobilized and used for the continuous production of l-(+)-tartaric acid.