The invention provides ulinastatin and a preparation method thereof. The absorbance value of 50,000 units/ml ulinastatin at 405nm is not more than 0.09, the content of human urinary kininogenase is not more than 0.00025PNAU, the content of heavy metal is not more than 0.8micro g/ml, and the content of bacterial endotoxin is not more than 3.0EU/ml. Urine is pretreated by methods such as nitrite, ammonium sulfate, polyethylene glycol, pH regulation and the like; preliminary purification is carried out by adopting a dialysis method before passing through a column; and the ulinastatin pure productis prepared sequentially through a gel column and a cation exchange column, so that the preparation process is greatly simplified, the purity and yield of the finished product are ensured, and the drug activity is greatly improved.