Objective To evaluate the in vitro effect of combinations of 5 β-lactam antibiotics with different β-lactamase inhibitors on the activity of multidrug-resistant Mycobacterium tuberculosis (MDR-TB), and identify the most effective combination of β-lactam antibiotics and β-lactamase inhibitors against MDR-TB. Methods MDR-TB strains collected in Henan Province Antimicrobial Resistance Surveillance Project in 2021 were selected. The minimum inhibitory concentrations (MIC) of 5 β-lactam antibiotics or combinations with different β-lactamase inhibitors on clinically isolated MDR-TB strains were measured by MIC detection method, and the blaC mutation of the strains was analyzed by polymerase chain reaction (PCR) and DNA sequencing. Results A total of 105 strains of MDR-TB were included in the analysis. MIC detection results showed that doripenem had the highest antibacterial activity against MDR-TB, with a MIC₅₀ of 16 μg/mL. MIC values of most β-lactam antibiotics decreased significantly after combined with β-lactamase inhibitors. A total of 13.33% (n=14) strains had mutations in blaC gene, mainly 3 nucleotide substitution mutations, namely AGT333AGG, AAC638ACC and ATC786ATT. BlaC proteins Ser111Arg and Asn213Thr enhanced the synergistic effect of clavulanic acid/sulbactam and meropenem on MDR-TB compared with synonymous single-nucleotide mutation. Conclusion The combination of doripenem and sulbactam has the strongest antibacterial activity against MDR-TB. Substitution mutations of BlaC protein Ser111Arg and Asn213Thr enhances the sensitivity of MDR-TB to meropenem through the synergy with clavulanic acid/sulbactam.