[Objective] Reteplase (rtPA, recombinant tissue plasminogen activator) is considered as the third generation of safe and effective thrombolytic agent. In order to explore the suitable system for secretory expression of rtPA, the rtPA gene was expressed in three different phenotypes of Pichia pastoris with pPIC9K as the vector. [Methods] The rtPA gene was obtained from plasmid pET28a-rtPA and inserted into the secretory expression vector pPIC9K. The recombinant plasmid pPIC9K-rtPA was digested with restriction endonuclease Sal I and then transformed into P. pastoris GS115, SMD1168 and KM71, respectively. The positive clones were screened on MD-G418 plates and verified by PCR. The expression of rtPA in the recombinant strains were induced with methanol, and analyzed by Western blot and fibrin agarose plate. [Results] The molecular weight of recombinant proteins was about 43 kD; both rtPA-GS115 and rtPA-KM71 had a specific band at 39 kD, and the former had a slight degradation band at 32 kD, while the latter did not; rtPA-SMD1168 showed no degradation. The specific fibrinolytic activity of rtPA-SMD1168 was 27% higher than that of rtPA-GS115, while the expression and activity of rtPA-KM71 displayed the lowest level among tested samples. [Conclusion] According to the expression of fibrinolytic activity, P. pastoris SMD1168 was proven to be the best expression system. On the premise of controlling the activity of protease and reducing the degradation of products, P. pastoris GS115 was selected as the preferred system for rtPA expression.