Objective To investigate the interaction between rifampin(RFP)and levofloxacin(LVFX)in retreatment of smear positive pulmonary tuberculosis(TB). Methods One hundred retreated smear positive TB patients admitted to our hospital between January 2013 and December 2015 were randomly divided into two groups:the control group in 50 patients with only RFP treatment,and the treatment group in 50 patients with both RFP and LVFX treatments. The RFP plasma concentration was tested 0.5 h,1 h,and 2 h after intake of medicine. Sputum smear test was performed at the end of February,May,August and September to investigate the interaction between RFP and LVFX in retreatment of smear positive TB. Results The plasma concentration of RFP did not show significant difference(P > 0.05)between the control group and the treatment group at 0.5 h,1 h,and 2 h after intake of medicine. As the treatment time went on,the level of smear positive gradually decreased. The sputum smear positive rate after 2,5,8,and 9 months of treatment showed significant difference between the control group and the treatment group. After 8 months of treatment,the ratio of stabilized patients and patients under treatment also showed significant difference(P<0.05). Conclusion Rifampicin and levofloxacin have no antagonism effect in retreatment of smear positive TB,and RFP and LVFX can be used together to treat the repeated smear positive TB.