Human urinary kallikrein (hk-1) and human urinary trypsin inhibitor (hUTI) are two acidic proteins in the urine. Both of them are important drugs. The way of colloid adsorption together with ethyl alcohol precipitation isolated these two proteins successfully. By using of ion-exchange, hydrophobic, affinity chromatography and gel filtration, human urinary kallikrein(hk-1) was purified with single band on SDS-PAGE and single peak on HPLC. The molecular mass was 33 450 u on MALDI-TOF-MS, the pI was about 4.3 on IEF. The influence of pH and temperature on the activity of hk-1 was studied. The whole procedure is suitable for large-scale production.