Objective: To establish size exclusion chromatography（SEC） and sodium dodecyl sulfate capillary electrophoresis（CE-SDS） for the determination of the molecular fractions in urokinase,and the methodological validation and sample determination results of the two methods were compared. Methods: SEC was performed on a TSKgel G2000SWXL column（7.8 mm×300 mm,5 μm） with 40.1 mol·L^-1 sodium dihydrogen phosphate buffer （pH 3.0） mobile phase as the mobile phase,the flow rate was 0.5 mL·min^-1,the column temperature was 35 ℃, the detection wavelength was 280 nm and the injection volume was 50 μL．CE-SDS was performed on a uncoated fused quartz capillary tube（50 μm×30.2 cm,effective length 20.2 cm）,the separation voltage was 15 kV,the column temperature was 25 ℃,the detection wavelength was 214 nm,the injection end was positive electrode and the injection pressure was 5 kV for 20 s. Results: By comparing the results of the methodological validation and sample determination results of the two methods,it was proved that the results of the two methods were almost the same．The SEC method results showed the linear relationship of high molecular mass and low molecular mass urokinase was good when the concentration of urokinase was in the range of 0.05-5.1 mg·mL^-1．The detection limit of high molecular mass and low molecular mass urokinase was 1.5 μg·mL^-1 and 5.1 μg·mL^-1,respectively,and the lower limit of quantification was 5.1 μg·mL^-1 and 16.9 μg·mL^-1,respectively．The relative content range of high molecular mass and low molecular mass urokinase was 87.0%-96.4% and 3.6%-13.0%,respectively．The CE-SDS method results showed the linear relationship of high molecular mass and low molecular mass urokinase was good when the concentration of urokinase was in the range of 0.1-5.2 mg·mL^-1．The detection limit of high molecular mass and low molecular mass urokinase was 2.5 μg·mL^-1 and 51.8 μg·mL^-1,respectively,and the lower limit of quantification was 7.5 μg·mL^-1 and 155.4 μg·mL^-1 respectively．The relative content range of high molecular mass and low molecular mass urokinase was 87.0%-96.4% and 3.6%-13.0%,respectively. Conclusion: Both methods are suitable for the determination of molecular fractions in urokinase and complement for each other.