Objective To study the distribution of fungal species in otomycosis and explore the method of multiplex PCR technology for detecting and identifying common fungal species in otomycosis. Methods External ear canal specimens were collected from 137 patients with otomycosis who visited the Otolaryngology Department of Jingzhou Central Hospital from March 2022 to June 2023. We utilized an improved Martin solid medium for routine cultivation of a portion of the same specimen. Following the cultivation, DNA was extracted, followed by PCR amplification and sequencing to identify the fungal species,and then were subjected to two times of multiple PCR, respectively using universal fungal primers and specific fungal primers. The fungal species could be identified according to the length of the bands obtained by agarose gel electrophoresis. Results Through traditional methods and multiplex PCR technology, a total of 128 strains (93.4%) of Aspergillus were identified in 137 patients with otomycosis in the local area, including 73 strains of Aspergillus terreus, 24 strains of Aspergillus flavus, 18 strains of Aspergillus niger, and 13 strains of Aspergillus fumigatus; There are a total of 9 strains (6.6%) of Candida, all of which are Candida albicans. The traditional method takes 4-7 days, while the multiplex PCR technology can identify fungal species within 6-8 hours. Comparing the results of the two methods, the conformity rate is 100%. Conclusion The most common fungal species in patients with otomycosis in this region are Aspergillus and Candida. In addition to common bacterial strains, multiplex PCR technology can also identify rare bacterial strains such as highly resistant and lethal Candida albicans and Candida mirabilis. Compared with traditional methods, multiplex PCR technology has the advantages of fast and accurate detection and identification of otomycosis, which is worthy of clinical promotion.