Objective To evaluate the application value of nucleic acid mass spectrometry in detecting ERG11 gene mutations associated with azole resistance in Candida tropicalis （C. tropicalis）， thereby providing a scientific basis for the rational clinical use of azole antifungal agents. Methods Clinical isolates of C. tropicalis were obtained from the Affiliated Hospital of Xuzhou Medical University between July 2023 and November 2024. For each isolate， specific ERG11 mutations （A395T and C461T） were analyzed using Sanger sequencing and nucleic acid mass spectrometry. Sanger sequencing was used as the reference standard to evaluate the performance of mass spectrometry in mutation detection. Results The established nucleic acid mass spectrometry method effectively identified four genotypes of ERG11 （A395T and C461T）， with distinct spectral peaks for each mutation and no cross-interference observed. Among 88 clinical isolates， the sensitivity， specificity， positive predictive value （PPV）， and negative predictive value （NPV） for the A395T mutation were 86.7%， 100%， 100%， and 97.3%， respectively. For the C461T mutation， the corresponding values were 81.3%， 100%， 100%， and 96.0%， respectively. Kappa statistics demonstrated a high level of agreement between mass spectrometry and sequencing results. Conclusions Nucleic acid mass spectrometry exhibits high sensitivity and specificity in the detection of ERG11 mutations， enabling rapid， accurate， and adaptable high-throughput analysis. This makes it a highly effective method for identifying mutations associated with fungal resistance.