Abstract: The fibrinolytic enzymes which wildly existed in many kinds of snake venoms have important medicine effect, it contributed to open up snake venom resources from gene engineering. Total RNA was extracted from gland of Gloydius intermedius snake venom, fibrinolytic enzyme (FLE) gene was amplified by RT-PCR. The gene was ligased with pMD18-T vector, then FLE was obtained; It was subcloned into pPROEXHTb prokaryotic expressive vector to construct expressive vector; The positive recombinant expression vector was transformed into E. coli and was induced to express recombinant protein FLE. The fusion protein was detected by SDS-PAGE. The results indicated that prokaryotic expression vector ppPROEXHTb-FLE was constructed successfully, and expressed in E. coli, the molecular weight of recombinant protein was about 30kD. It was effective to use the prokaryotic expressive vector to express. All that would provided base for gene engineering products of snake venom FLE in the future.