A procedure was developed for detecting heparinase activity on heparin agar plates. The method is based on the differential precipitation of heparin and heparinase-generated heparin fragments by protamine sulfate. Heparinase activity is detected by the presence of clear zones against a white background. This method can be used to screen for the expression of recombinant heparinase and to identify Flavobacterium heparinum mutants expressing heparinase constitutively.