To develop a purification process using hollow fiber membrane separation combined with size exclusion chromatography for the extraction of lumbrokinase from earthworms (Lumbricus rubellus). To extract the protein, the earthworms were first homogenized for 10 min, to produce ultrafine particles. Polyether sulfone hollow fiber membranes with MW cut offs of 50 and 6 kDa were used for initial purification of the crude extract. Further purification was carried out on a Sephadex G-75 column, and yielded three fractions of high purity protein. One of these fractions showed fibrinolytic activity. Three samples of high purity protein were obtained and one protein (LK1) showed strong fibrinolytic activity. The method has higher purification efficiency in comparison with existing methods.