The invention relates to a culture medium suitable for clostridium difficile, and a preparation method thereof, and belongs to the technical field of microbial cultivation. The preparation method is used for solving a problem in the prior art that culturing effect of conventional culture medium on clostridium difficile is poor. The culture medium comprises composite ingredients such as peptone and dehydrated beef heart infusion, and also comprises complement components such as glycine and laminine. It is shown by experiment results that culture effect of the culture medium on clostridium difficile is excellent, and the culture medium possesses important implication in study on the biological characteristics of clostridium difficile and on treatment of clostridium difficile infection.