The invention relates to the technical field of detection, in particular to a dairy cow mastitis detection kit which comprises a gram-negative bacterium chromogenic culture medium, a gram-positive bacterium chromogenic culture medium and an enterococcus-staphylococcus aureus co-chromogenic culture medium. The gram-negative bacterium chromogenic culture medium comprises a first basic culture medium and a first beef powder medium, the gram-positive bacterium chromogenic culture medium comprises a second basic culture medium, a second beef powder medium and sodium chloride, and the enterococcus-staphylococcus aureus co-chromogenic culture medium comprises an enterococcus basic culture medium and a staphylococcus aureus basic culture medium. The dairy cow mastitis detection kit can quickly and accurately detect recessive dairy cow mastitis, results can be directly judged by naked eyes without the aid of special instruments, technical personnel can master quite easily, operation is simple and convenient, a result can be acquired within 18-24h, and the kit is supportive of mass detection and low in cost.