Abstract: Objective To analyze drug susceptibility of multi-drug resistant (MDR) Mycobacterium tuberculosis isolated from Ningbo, Zhejiang province, and provide scientific evidence for MDR-tuberculosis (MDR-TB) control and prevention. Methods A total of 116 MDR-TB isolates were selected from the first drug resistance survey in Ningbo during 2014-2015. The conventional drug susceptibility test was conducted to detect the strains' drug-resistance to 8 second-line anti-TB drugs (levofloxacin, ofloxacin, moxifloxacin, kanamycin, amikacin, capreomycin, protionamide and 4-Amino-2-hydroxybenzoicacid). The drug-resistant rate and cross resistance were analyzed. Results For second-line anti TB drugs, the drug-resistant rates to levofloxacin, ofloxacin, moxifloxacin, kanamycin, amikacin, capreomycin, protionamide and 4-Amino-2-hydroxybenzoicacid were 31.0%, 29.3%, 9.4%, 8.6%, 7.8%, 3.4%, 6.0% and 19.8%, respectively. The drug-resistant rates to capreomycin in new case group were significantly higher than that in re-treated case group (2=4.925, P0.05). In addition, there were the cross-resistances to kanamycin and amikacin, toofloxacin and levofloxacin. Conclusion The resistance rate to second-line anti-TB drugs was high and extensive drug resistance was also detected in TB patients in Ningbo, suggesting that it is necessary to conduct the survey of TB strain's susceptibility to second line anti TB drugs for the rational treatment of TB.