Objective To evaluate the clinical application value of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in analyzing the homology of Acinetobacter baumannii (AB). Methods After excluding repetitive strains from multiple specimens of the same patient or environment, a total of 46 AB strains isolated from patients' sputum and environmental specimens of neurological intensive care unit (ICU) in a tertiary first-class general hospital from May 2020 to February 2021 were collected. Strains were detected by VITEK-MS mass spectrometer. Cluster analysis was performed by SARAMIS Premium software, and verified by multilocus sequence typing (MLST). Results Cluster analysis and comparison of MALDI-TOF MS and MLST found that among the 46 AB strains, 39 were the type MS-a of MALDI-TOF MS, of which 22 strains were the cluster MT-A of MLST, including ST208 (n=3), ST540 (n=3), ST195 (n=8), ST369 (n=5), ST136 (n=1), ST436 (n=1) and ST1893 (n=1); 16 strains were MT-B, including type ST381(n=4), type ST469 (n=11), and type ST938 (n=1); one strain was cluster MT-C (ST1821); one strain of type MS-b was ST381; two strains of type MS-c were ST369; one strain of type MS-d was ST195; two strains of type MS-e were ST540 and ST369, respectively; one strain of type MS-f was STN1. Conclusion As a homology analysis method, MALDI-TOF MS still has certain limitations such as low consistency with MLST results, low resolution and specificity, thus cannot replace MLST technology.