The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely applied in routine clinical microbiology laboratories as an efficient and reliable technique for diagnostic purpose. In this work, we evaluated the performance of the newly developed Zybio EXS3000 (Zybio Inc., China) in microbial identification and compared it with VITEK MS (bioMérieux, France). For this study, a total of 1340 isolates from various clinical specimens were collected. These isolates were analyzed simultaneously on both EXS3000 and VITEK MS. The inconsistent or unidentifiable data were further identified using the help of either 16S rRNA gene or ITS region sequencing. During the study, we observed that EXS3000 and VITEK MS provided positive confirmatory diagnostics for 95.0% and 96.5% of the isolates, respectively, which were consistent with the sequencing results. However, it is worth noting that the EXS3000 system needs to improve the identification performance of Candida albicans in the follow-up. There are no significant differences between the two devices in terms of microbial identification performance. The advantage of EXS3000 over VITEK MS is in its ability to perform in significantly lesser time period. In conclusion, the results of this investigation showed that EXS3000 can be used to identify microorganisms in clinical microbiology laboratories.© 2023 The Authors.