Abstract: [Objective] To explore the preparation method of commonly employed antimicrobial susceptibility disk for veterinary use and to saving the cost of antimicrobial susceptibility test. [ Methods] According to three concentration gradients of treatment dosage （marking l×）, twice treatment dosage （marking 2×） and four times treatment dosage （marking 4×）, six kinds of antibiotics commonly used in veterinary clinic including penicillin G, kanamycin, gentamicin, ofloxacin, streptomycin and ceftiofur sodium were selected to prepare antimicrobial susceptibility disk. The antimicrobial susceptibility test was carried out with the self-prepared disks and the commercially available disks, respectively, and the antimicrobial effect of them were compared. [ Results] Expect for ceftiofur sodium, the other five kinds of disks containing twice treatment dosage were consistent with the commercially available disks. [Conclusion] The method for preparing antimicrobial susceptibility disk for veterinary use developed in this study was successful.