A novel plasminogen activator containing low molecular single-chain urokinase (scuPA-32k) and fibrin β chain polypeptide (Fβ 15～42) was designed and constructed. ScuPA-32k cDNA was obtained by polymerase chain reaction (PCR) from pro-urokinase gene; while Fβ (15～42) cDNA was generated by joining synthesized oligonucleotide fragments together. Through suitable linker and approximately restriction site, scuPA-32k and Fβ (15～42) cDNA were ligated together. The fusion protein was expressed by IPTG induced in E.coli. After denaturation and renaturation, the aim protein was purified to homogeneity by Zn²⁺ chelating chromatography and Sephacryl S200 chromatography. The apparent molecular mass was 35 ku shown by SDS-PAGE analysis. The special activity was 87 000 U/mg detected by fibrin plate determination. The enzyme had similar kinetic parameters to that of natural uPA-32k when was assayed with the chromognic substrate S2444. However Fβ(15～42)/scuPA-32k had higher fibrin affinity than that of natural scuPA-32k and had antifibrin polymerization. These results showed that the fusion protein had good respects.