Objective To explore the clinical significance of fluorescence staining in the rapid diagnosis of fungal otitis externa. Methods A retrospective analysis was performed on 30 children with suspected fungal otitis externa, who were diagnosed at the Otolaryngology Outpatient Clinic of Xiamen Children’s Hospital from January 2021 to December 2023. All cases were examined by external ear canal secretion fluorescence staining and fungal cultivation. The positive rate of fungal infection was compared between fluorescence staining and fungal cultivation. Results Of the 30 suspected children, 28 cases were detected as fungal infections by fluorescence staining, and 26 cases were detected as fungal infections by fungal culture. The positive rate of fungal otitis externa was higher by fluorescence staining (93.3%) than by fungal cultivation method (86.7%), and the difference was statistically significant(χ²=0.04,P<0.05). Conclusion The fluorescence staining method is high detection rate in diagnosing fungal otitis externa. And the operation is more simple and fast. It is worthy of clinical promotion and application.