Objective　Recombinant human pro-urokinase forms insoluble inclusion body when overexpressed in Escherichia coli. It must be denatured and renatured in vitro so that it can acquire activity. This study aimed at increasing the renaturation yield of denaturant pro-urokinase. Methods　We evaluated the basic renaturation conditions of pro-urokinase through qualitative and quantitative analysis of pH, temperature, denatured concentration, protein concentration, and the ratio of reduced and oxidized thiol reagents. We also compared the effects of nonspecific additives, step-wise dilution and urea gradient dialysis. Results　We defined the optimal conditions of pro-urokinase renaturation with a yield of about 20%-30%. Conclusion　Different recombinant denatured proteins have different renaturation conditions due to their different molecular sizes, molecular constructions, disulfide bond numbers, and hydrophobicity. The renaturation yield can be increased by optimizing the renaturation conditions of a specific protein.