Aim: To develop emerging diagnostic technique for bovine tuberculosis and to identify its potential risk factors. Materials & methods: Bacterial genomic DNA was isolated from bovine milk and human sputum samples and subjected to PCR using specific primer pairs. PCR results were validated using bacteriological cultures. Results: PCR amplification of the targeted DNA fragment of Mycobacterium bovis was successful in 12.33% (37/300) of the bovine samples. Interestingly, 500-bp DNA fragment was also amplified in 6.67% (6/90) of the sputum indicating the possibility of zoonotic transmission. Rearing of livestock in household, unpasteurized milk consumption and smoking were identified as potential risk factors. Conclusion: Results of the study may add value to bovine tuberculosis eradication campaigns to achieve the One Health initiative.