Abstract: Objective: To evaluate the accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying clinical filamentous fungi. Methods: A self-built database was constructed on the basis of the original commercial reference database using AUTOF MS1000 mass spectrometry system, and 125 clinically isolated strains were used to verify it. Results: The self-built database newly added 15 species from 3 genera of dermatophytes, totaling 76 strains and 30 species of Aspergillus, totaling 75 strains.The genus-level identification rate of 125 clinically isolated strains was 100% based on existing commercial database and self-built database; the species-level identification rate reached 80.8%. Using the molecular identification as the gold standard, the correct identification rate was 94.1%. Among them, the species-level identification rate of dermatophytes was 79.7%, and the correct identification rate was 92.7%; the species-level identification rate of 56 Aspergillus was 82.1%, and the correct identification rate was 95.6%. Six strains were incorrectly identified, concentrated in T. mentagrophytes series whose T. interdigitale, T. mentagrophytes, and T. quinckeanum were cross-identification, and in Sect.Nigri whose A. niger and A. tubingensis were cross-identification. Conclusion: The accuracy of MALDI-TOF MS identification of filamentous fungi can be improved by supplementing and perfecting the self-built database. Meanwhile, the reference database should be expanded and updated in time to ensure the ability of MALDI-TOF MS to accurately identify fungal strains.