Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors， including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers， limitations to developing an effective matriptase inhibitor include weak binding affinity， poor selectivity， and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1)， a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain， whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here， we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a of 70 ± 5 pm， an increase of 120-fold compared with the natural HAI-1 inhibitor， to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore， the engineered inhibitor demonstrates a protease selectivity profile similar to that of wildtype KD1 but distinct from that of HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1.