Abstract: Objective：To evaluate the application of novel fluorescent staining method with hypersensitivity and enhancement for microscopic examination of superficial fungal. Methods：A total of 200 cases with clinically suspected superficial fungal infection were screened by the hypersensitive, enhanced fluorescent staining, calcofluor white staining and microscopic examination with 1.78 mol/L potassium hydroxide (KOH) based smear assay, respectively. The positive percentages of the different methods were calculated and compared. Results：In the developed fluorescent staining examination, clean background was shown in the view field. The hyphae and spores displayed in bright blue, thus fungal morphology was so distinct that they were easy to be distinguished. Compared with calcofluor white staining and KOH based smear, the fluorescent staining showed high positive rate with significant difference respectively (χ²=17.984, P<0.05; χ²=32.063, P<0.05). Conclusion：The novel fluorescent staining should be a rapid, accurate method for microscopic examination of fungi, which is worthy to be widely used in clinical laboratories for early diagnosis of fungal infection.