Recombinant proteins were often expressed with His-tag to simplify the purification process. Among them, transaminase was mostly expressed with fusion tags and widely used in the production of numerous amino moieties. However, the existence of the His-tag has been reported to affect various properties of different recombinant enzymes, while the effect on transaminase was rarely studied. In this paper, we investigated the effect of His-tag on transaminase based on the various activities of 4-aminobutyrate-2-oxoglutarate transaminase (GabT) when it was expressed in vector pETDuet-1. We found that His-tag did not affect the enantioselectivity, but decreased the catalytic activity to different extents according to its existence and location. Native GabT maintained the highest catalytic activity; GabT with C-terminal His-tag showed slightly lower activity than native GabT but about 2.2-fold higher than GabT with N-terminal His-tag. Besides, other fusion tags like T7-tag and S-tag inserted between N-His-tag and GabT can relieve the decreasing effect of His-tag on GabT activity. Furthermore, whole cell catalytic activity of several transaminases was improved by deleting the N-terminal His-tag. This study provided a strategy for the efficient expression of recombinant transaminase with improved catalytic activity and might attract attention to the effect of His-tag on other enzymatic properties.