Objective To establish a guinea pig model for diagnostic reagent of tuberculosis. Methods By single or multiple subcutaneous injection of heat-killed H₃₇Rv in different doses in the groin of guinea pigs to establish a model of positive response to 0.1 mL (5 IU) standard tuberculin (TB-PPD) skin test. Results Three doses of heat-killed H₃₇Rv (0.2 mg/mL,0.3 mg/mL and 0.5 mg/mL) could be used to generate the model of biological diagnosis of tuberculosis. After 24 and 48 hours, the diameter of red spot by TB-PPD skin test was 15.4±2.3 mm when a dose of 0.2 mg/mL heat-killed H₃₇Rv was administered for immunizing and allergizing the guinea pigs. The biggest red spot was induced at doses of 0.3 mg/mL and 0.5 mg/mL. The test results showed that the immune response induced by multiple njection to immunizing and allergizing guinea pigs was not significantly different than that induced by single immunizing injection, and the first skin test was better than the second, third and fourth skin test (P≤0.05). In addition, the body weight of the guinea pigs was still increasing after infection with heat-killed H₃₇Rv, and ulcers occurred in the injection sites in some guinea pigs. Conclusions A single subcutaneous injection of 0.2 mg/mL heat-killed H₃₇Rv in guinea pigs can be used well to establish a reliable model for biological diagnostic reagent of tuberculosis. Increasing the sensitizing dose and multiple sensitization can not increase the intensity of the delayed-type hypersensitivity (DTH) response.