Enteropeptidase is a duodenum serine protease that triggers the activation of pancreatic enzymes by remarkably specific cleavages after lysine residues of peptidyl substrate (Asp)₄-Lys. This high specific cleavage makes the enzyme a widely used biotechnological tool in laboratory researches and industrial scale. Previous studies both in small and large scales were showed low expression and miss-folding of the expressed protein. In this study, the DNA sequence encoding the light chain (catalytic subunit) of bovine enteropeptidase (EP_L) was subcloned into plasmid pET-32b, downstream to the DNA encoding the fusion partner thioredoxin immediately after the EP_L cleavage site. SHuffle® T7 Express was selected as an expression host due to the ability to promote proper folding and correction of the mis-oxidized bonds. Expression and purification of protein was performed, and the result of biological activity confirmed that the active EP_L was obtained. Optimization of protein expression conditions was accomplished by response surface methodology for significant factors including induction temperature, duration of induction, inducer concentration and OD₆₀₀ of induction. The best conditions were achieved in 1.05 mM IPTG at OD₆₀₀ of 0.6 for seven h incubation at 26.5 °C, and a high level of protein expression was obtained in the optimized condition.