Abstract: Human placental hyaluronidase preparation is prepared by extracting hyaluronidase from human placenta with an aqueous alkaline solution, harvesting globulin fraction from the extract, selectively adsorbing hyaluronidase contained in the fraction on an anion exchanger such as DEAE-Cellulose and eluting it, gel-filtrating the eluate, and lyophilizing the filtrate into powder. The hyaluronidase thus obtained has a molecular weight of about 70,000, with isoelectric point of pH 5.2, and enzymatic optical pH ranging 3.6 - 4.0, differing from conventional bovin testicular hyaluronidase. The preparation contains no heterogeneous proteins which cause ill-effect to human body.