Abstract: Objective To evaluate the application value of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) technique in the rapid identification of rare non-fermentative bacteria. Methods From July 2013 to October 2016, 67 clinical isolates of rare non-fermentative bacteria were collected from the First Affiliated Hospital of Anhui Medical University, which included 21 strains isolated from sputum, 13 strains from urine, 9 strains from blood, 9 strains from bone marrow, 8 strains from secretions, 2 strains from peritoneal dialysis fluid, 1 strain from cerebrospinal fluid, 1 strain from bile, and 3 strains from other body fluids. According to the gold standard of 16SrRNA sequence analysis, the accuracy of the identification system of MALDI-TOF MS and Vitek2 Compact was compared. Results In the 67 strains of bacteria, MALDI-TOF MS technique identified 66 strains (98.5%) to species level and 1 strains (1.5%) to genus level; while Vitek2 Compact identified 59 strains (88.1%) to species level, and 8 strains (11.9%) failed to identify. 16SrRNA gene fragments were amplified from the 67 strains of experimental bacteria and successfully sequenced, all of the strains were identified to species level. Using 16SrRNA sequence analysis as the gold standard, the accuracy of MALDI-TOF MS system and Vitek2 Compact system for identification at the species level was 98.5% (66/67) and 88.1% (59/67) respectively. Conclusions Compared with traditional bacteria biochemical reaction method, MALDI-TOF MS technique is simple, rapid and inexpensive. Its accuracy of identification result is highly consistent with that of 16SrRNA sequence analysis, MALDI-TOF MS technique can improve the accuracy of clinical identification of non-fermentative bacteria.