Objective To investigate the rapid identification of bla_KPC-2-harboring Klebsiella pneumoniae (K. pneumoniae) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods K. pneumoniae isolated from hospitalized patients in the First Affiliated Hospital of Bengbu Medical College from September 2018 to November 2020 were collected, antimicrobial susceptibility testing was performed by disk diffusion method. bla_KPC-2-harboring K. pneumoniae was screened by polymerase chain reaction (PCR). MALDI-TOF MS was used to identify the strains, the mass spectra of bla_KPC-2-harboring K. pneumoniae and carbapenems-sensitive K. pneumoniae (CSKP) were collected, 70 strains were selected respectively to establish the Super-Spectra of bla_KPC-2-harboring K. pneumoniae and CSKP. K. pneumoniae except strains in Super-Spectra were also identified for judging whether the identification results were accurate according to antimicrobial resistance phenotype and PCR results. Results A total of 143 carbapenems-resistant K. pneumoniae (CRKP) strains and 152 CSKP strains were screened out from 295 K. pneumoniae strains. 140 CRKP strains were identified carrying carbapenemase gene (134, 3, 2, and 1 strains were found bla_KPC-2, bla_KPC-18, bla_NDM-1 and bla_IMP respectively), 3 strains didn't harbor carbapenemase gene, 2 strains were found both bla_KPC-2 gene and bla_NDM-1 gene. According to the require-ment of database establishment, a Super-Spectra of bla_KPC-2-harboring genotypes of K. pneumoniae and CSKP were established; the coincidence rate of the two was 80% when the error value was < 0.5; according to the comparison map, the five peaks of 4 154.4m/z, 8 310.7m/z, 10 880.8m/z, 3 579m/z and 10 079.3m/z could be used as the characteristic peaks to distinguish the bla_KPC-2-harboring K. pneumoniae and CSKP. 155 strains of K. pneumoniae except Super-Spectra were selected for verification, the accuracy rate was 92.90% (144/155); the accuracy of bla_KPC-2-harboring K. pneumoniae was 94.52% (69/73), and that of CSKP was 91.46% (75/82). Conclusion The establishment of Super-Spectra through MALDI-TOF MS can rapidly predict bla_KPC-2 genotype K. pneumoniae, which is helpful to provide a reliable laboratory basis for the diagnosis and treatment of CRKP infection as well as prevention and control of healthcare-associated infection.