It was found that the serine protease inhibitors BmSPI38 and BmSPI39 in silkworm can strongly inhibit the activity of porcine pancreatic elastase, which has potential applicational value in the drug research and development of lung diseases, inflammatory diseases, and skin aging caused by the excessive release of elastase. Previous studies have shown that homotypic multimers obtained by tandem expression can significantly enhance the antifungal activity and structural homogeneity of BmSPI38 and BmSPI39, while the effect of the tandem expression of these two inhibitors, with different combinations, on the total activity and expression levels of multimers remains unclear. The aim of this study is to explore whether it is possible to obtain the combination of BmSPI38 and BmSPI39 with strong total expression activity by protein engineering. In this study, 40 tandem multimer expression vectors with different combinatorial forms of BmSPI38 and BmSPI39 were constructed by the isocaudomer method, and recombinant proteins were obtained by the prokaryotic expression system. The target proteins were separated by SDS-PAGE to analyze the expression levels of multimer proteins with different combinatorial forms. The total activity of the recombinant expression products with different tandem forms was investigated using the in-gel activity staining technique of protease inhibitors. The SDS-PAGE results show that the expression levels of tandem multimers containing the BmSPI39 module at the carboxyl terminus were generally higher in the