Tissue plasminogen activator (tPA) a thrombolytic agent is commonly used for digesting the blood clot. tPA half-life is low (4-6 min) and its administration needs a prolonged continuous infusion. Improving tPA half-life could reduce enzyme dosage and enhance patient compliance. Nano-carries could be used as delivery systems for the protection of enzymes physically, enhancing half-life and increasing the stability of them. In this study, chitosan (CS) and polyethylene glycol (PEG) were used for the preparation of CS-g-PEG/tPA nanoparticles (NPs) via the ion gelation method. Particles' size and loading capacity were optimised by central composite design. Then, NPs cytotoxicity, release profile, enzyme activity and in vivo half-life and coagulation time were investigated. The results showed that NPs does not have significant cytotoxicity. Release study revealed that a burst effect happened in the first 5 min and resulted in releasing 30% of tPA. Loading tPA in NPs could decrease 25% of its activity but the half-life of it increases in comparison to free tPA in vivo. Also, blood coagulation time has significantly affected (p-value = 0.041) by encapsulated tPA in comparison to free tPA. So, CS-g-PEG/tPA could increase enzyme half-life during the time and could be used as a non-toxic candidate delivery system for tPA.