The invention provides a method for preparing heparinase I. The method for preparing the heparinase I comprises the following steps of: inoculating flavobacterium heparinum serving as a raw material to a seed culture medium for culture; then inoculating the flavobacterium heparinum to a fermentation culture medium; centrifugally collecting precipitate; performing ultrasonication on the precipitate; performing centrifugation again to obtain crude enzyme liquid of the flavobacterium heparinum heparinase I; and performing SP-sepharose FF chromatographic purification on the crude enzyme liquid for three times to obtain the high-purity flavobacterium heparinum heparinase I, wherein the SP-sepharose FF chromatographic purification for three times is protected by calcium chloride in the whole course, so that the yield of pure enzymic activity is greatly increased. The method for preparing the heparinase I has the characteristics of simple process, easy amplification, large preparation amount of products at a time, low cost of reagents and the like. The specific activity of the prepared heparinase I reaches 223 IU/mg and the yield of the pure enzymic activity reaches 30 percent. Compared with the conventional newest method, the method for preparing the flavobacterium heparinum heparinase I has the advantages of increasing the specific activity by more than two times, and the purification yield by about one time.