Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis.