Abstract: Objective To evaluate the cut-off value for identification of clinical yeasts with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS),aiming to provide more optimized value. Methods Totally 494 yeasts were isolated from clinical samples.We smeared a small amount of the yeast from a colony directly onto a target plate and covered it with 1μl of formic acid,and then the MALDI-TOF MS identification was performed and the result was recorded.The identifications of all strains were verified by sequence analyses of 18srDNA.ROC curve analysis was used to calculate the cut-off value,and the accuracy evaluated by new cut-off score and the manufacturer-recommended cut-off score was compared. Results The identification results of 27 species,463 strains of yeasts were consistent with sequencing result,with the accuracy of 93.7%.The cut-off value of 1.6 showed the largest area under the ROC curve,with a sensitivity of 0.86 and a specificity of 0.88,which was considered as the most optimal cut-off value.Compared with cut-off value of 2.0,the accuracy rate increased to 77.7% with the cut-off value of 1.6,and the difference was statistically significant (P<0.001). Conclusion Determinating and applying the optimal cut-off value will be helpful to improve the identification ability of clinical yeasts with MALDI-TOF MS.