A rapid serological test for tuberculosis (TB) infection was designed using antigens specific to Mycobacterium tuberculosis. Tuberculosis infection, TB vaccination and exposure to environmental Mycobacteria cannot be distinguished using skin tests based on tuberculin protein derivatives. The standard diagnostic techniques such as skin tests, X-rays and DNA techniques are time consuming, expensive, and not practical for screening large populations. We used the 38, 63, 64, 14, 59-kDa antigens of M. tuberculosis to develop a rapid immunochromatographic test kit. This study evaluates the diagnostic potential of the rapid test kit using TB positive and TB negative serum samples from various hospitals in India. The samples were obtained from patients infected with or exposed to bacteria and viral pathogens. The results demonstrated that the combination of antigens improved the diagnostic specificity and sensitivity. The specificity of the test was 99.42% with sensitivity of 98.52% (n = 241). In case of multiple infections, the specificity was 93.15% with a low sensitivity of 73.52% n = 141). The test kit may offer an improved alternative to purified protein derivative (PPD). This rapid TB test kit may be a useful tool for first-line testing of suspected cases, epidemiological studies and in designing a quality health system to reduce health hazards in resource-poor countries.