An object of the present invention is to provide a pharmaceutical composition comprising an enzyme-treated human serum which is useful for treatment and prevention of diseases such as a cancer and an infectious disease, and a method of preparing the same. The present invention relates to a method of preparing a pharmaceutical composition comprising an enzyme-treated human serum, comprising a step of bringing the human serum into contact with β-galactosidase and, to a pharmaceutical composition comprising an enzyme-treated human serum obtained by the preparation method.