Highly purified urokinase is obtained in a high yield by adsorbing urokinase from an aqueous crude urokuinase solution including human urine at a pH of about 4.0 to about 7.0 on a water-insoluble polysaccharide containing agarose and agaropectin, for example, agar and Sepharose, and by eluting urokinase with an alkaline solution (pH of about 8 to 11) or concentrated salt solution.