The invention belongs to the technical field of microbial culture, and concretely relates to an anaerobic enrichment medium and a preparation method thereof. The medium includes peptone, yeast extract powder, a brain and heart infusion broth, glucose, sodium thioglycolate, hemin, vitamin K1, an anticoagulant, an anaerobic indicator and distilled water; and the medium for promoting mass propagation of anaerobic bacteria can be prepared through a special ratio of the above components. The medium is used to clinically culture anaerobic pathogens in order to improve the positive detection rate of the anaerobic pathogens.