Objective To evaluate the application value of matrix-assisted laser desorption ionization time-of- ?ight mass spectrometry (MALDI-TOF MS) in the rapid detection of carbapenem-resistant enterobacteriaceae (CRE). Methods A total of 104 suspected CRE strains were collected from January 2023 to December 2024. PCR detection of genes such as bla_KPC, bla_NDM, bla_VIM, and bla_IMP was compared with MALDI-TOF MS results. Characteristic mass spectrometry peaks were collected and analyzed, and receiver operating characteristic (ROC) curves were constructed to evaluate diagnostic performance. Results PCR detected 73 carbapenemase-positive strains (70.19%). The diagnostic sensitivity and speci?city of the MALDI-TOF MS method were 92.31% and 95.12%, respectively. The area under the ROC curve (AUC) reached 0.880, with a Kappa value of 0.873 (P < 0.001), indicating high concordance with PCR results. Conclusion MALDI-TOF MS enables rapid and accurate identi?cation ofCRE, o?ering advantages such as ease of operation, high speci?city, and timely results. This provides a detection pathway for early screening of CRE and early warning of resistance mechanisms.