The aim of this study was comparative analysis of single intradermal comparative cervical tuberculin test (SICCT), immuno-chromatography analysis and polymerase chain reaction for diagnosis of tuberculosis. During the period from January-2019 to February-2020, a total of 800 animals (620 cattle & 180 buffalo) from 20 farms were screened by SICCT test in the different region of Gujarat state. For comparative study between SICCT, LFA and PCR, a total of 40 animals (11 SICCT positive, 09 SICCT negative, 10 Avian positive (PPD-A) and 10 SIT (PPD-B) positive animals) were selected. For PCR, additionally 10 lungs (02 deer and 08 cattle) samples suspected for bTB were also collected from the dead animals. A total of 106 samples comprised of 40 serums for LFA and 56 samples (16 milks, 40 nasal swabs and 10 lungs) were collected for PCR. Prevalence of bovine TB in the cattle and buffalo was 1.37% by SICCT test and 10.62% by SIT test was observed in the Gujarat state. The LFA contains the purified recombinant M. bovis specific antigens, all the 40 sera samples were tested by LFA and 08 (20.00%) animals were found to be positive for presence of antibody against M. bovis. Out of 40 SICCT selected animals, 23 (57.50%) were found positive in PCR using IS6110 (present in all MTC). Out of 23 MTC positive animals, species-wise, one M. bovis and eight M. tuberculosis or other members identified using targeting RD4 and RD9 regions. Hence, PCR could detect a greater number of animals as compared to SICCT test and LFA. Sensitivity of PCR was 100.00% and 85.71% when compared with SICCT test and SIT test, respectively. Sensitivity of LFA was 54.54% and 38.09% when compared with SICCT test and SIT test, respectively. PCR was found to be more sensitive and less specific than LFA when compared with tuberculin skin test (SICCT or SIT).