The gene fragment of urokinase catalytic domain mutant (C279A/N302Q) was amplified by the site-mutated PCR method and was cloned into pPICZαA secretory expression plasmid. The recombinant plasmid was transformed into yeast X-33 and selected with Zeocin. The recombinant protein was captured by the cation exchange chromatography SPFF and was purified to 99% of purity. The recombinant mutant protein was crystallized by the method of sitting-drop vapor diffusion. These crystals diffracted to 1.45 Å with synchrotron X-ray.