Abstract: Objective To understand the resistance of Mycobacterium tuberculosis to ofloxacin in Hangzhou and the characteristics of gyrA mutation in ofloxacin resistant M. tuberculosis. Methods The clinical isolates of M. tuberculosis were collected from every TB hospitals in Hangzhou from April 2010 to June 2012 to test their susceptibility to first-line drug, ofloxacin and kanamycin. The sequence of the quinolone resistance-determining region (QRDR) in gyrA gene of the ofloxacin resistant isolates was detected. Results A total of 1242 M. tuberculosis isolates were detected, in which 67 (5.39%) were resistant to ofloxacin. Among the 67 strains, 32 (47.76%) were resistant only to ofloxacin strains, 23 were multi-drug-resistant, accounting for 22.77% (23/101) of total multi-drug-resistant strains. Fifty five genomic DNA samples from the ofloxacin resistant strains were selected for gyrA gene sequence analysis. Results showed that gyrA mutation was found in 33 isolates (60%), in which 27.27% were at codon 90 and 32.73% were at codon 94. Conclusion The resistance to ofloxacin was high both in mono-drug-resistant TB and multi-drug-resistant TB patients in Hangzhou. It is necessary to strengthen the early detection of TB patients and the management of clinical antibiotic use.