Fusion expression is widely employed to enhance the solubility of recombinant proteins. However, removal of the fusion tag is often required due to its potential impact on the structure and activity of passenger proteins. Tobacco etch virus protease (TEV) is widely used for this purpose due to its stringent sequence recognition. In the present work, fusing to acyl carrier protein (ACP) significantly increased the yield of recombinant soluble TEV, and the ACP tag also greatly improved TEV stability. The cleavage activity of TEV was not affected by the ACP fusion tag, and ACP-TEV retained high activity, even at unfavourable pH. Moreover, ACP-TEV could be efficiently modified by co-expressed holo-ACP synthase (AcpS), leading to covalent attachment of the 4’-phosphopantetheine (4’-PP) group to ACP. The long, flexible 4’-PP (sulfhydryl group) displayed high specific reactivity with iodoacetyl groups on the solid support. Thus, TEV could be immobilised effectively and conveniently via the active holo-ACP, and immobilized TEV retained high cleavage activity after a long storage period and several cycles of reuse. As a low-cost and recyclable biocatalyst, TEV immobilised by this method holds promise for biotechnological research and development.