Reteplase (r-PA), as a plasminogen activator, has shown thrombolytic efficacy for thrombosis diseases. However, due to its short circulation time, double-bolus intravenous injection is required to maintain blood drug level. Improper dosing would bring about serious bleeding risk. Here we report a Polyhemoglobin-rPA preparation by introducing polymerized bovine hemoglobin (PolyHb) to crosslink r-PA with bifunctional reagent glutaraldehyde. Modified r-PA is more stable and possesses longer circulation time after intravenous injection. Results show that after intermolecular polymerization with hemoglobin, circulation half life span of r-PA was increased more than 65%. In vivo study demonstrated that, PolyHb-rPA significantly attenuated the severity of mice hemiparalysis and effectively recanalize rat embolism model. PolyHb-rPA could lead to r-PA administration dosage reduction, sustaining blood drug level, and bleeding risk decrease, which shows promise for its potential role as a protective therapeutic agent in clinical situations of myocardial infarction, cerebral thrombosis and other cardiovascular disease