Objective:To analyze the feasibility of fluorescence staining in the detection of Demodex mites. Methods:A single-center split-face study was conducted, and patients with clinically diagnosed rosacea or seborrheic dermatitis were enrolled from the Department of Dermatology, Southern University of Science and Technology Hospital from October 2020 to June 2021. Samples were obtained from the patients′ cheeks by using the squeeze-adhesion method, and Demodex mites were detected by fluorescence staining and direct microscopic examination separately. The detection rate, number of detected Demodex mites, and time for reading slides were compared between the above two detection methods, and the detection rate and number of detected Demodex mites were further compared between the fluorescence staining and standardized skin surface biopsy (SSSB). Chi-square test was used to compare enumeration data, and paired t-test for measurement data. Results:A total of 433 volunteers aged 28.3 ± 3.5 years were enrolled, including 185 males and 248 females. The performance of fluorescence staining was compared with that of direct microscopic examination in 338 pairs of samples obtained by the squeeze-adhesion method, and compared with that of SSSB in 95 pairs of samples obtained by the squeeze-adhesion method. The detection rate of Demodex mites by fluorescence staining was significantly higher (34.0%, 115/338) than that by direct microscopic examination (31.4%, 106/338; McNemar test, P = 0.004) ; among 118 positive samples, the number of detected Demodex mites by fluorescence staining ([8.0 ± 3.3]/cm 2) was also significantly higher than that by direct microscopic examination ([5.5 ± 2.9]/cm 2, t = 9.21, P < 0.001) ; the time for reading slides undergoing fluorescence staining was significantly shorter (8.3 ± 1.2 minutes) than that undergoing direct microscopic examination (17.3 ± 2.5 minutes, t = 38.44, P < 0.001) ; there was favorable consistency in fluorescence staining results between two clinical laboratorians (kappa value = 0.935, P < 0.001). The detection rate of Demodex mites by fluorescence staining (34.7%, 33/95) was higher than that by SSSB (33.7%, 32/95; McNemar test, P < 0.001) ; among 35 positive samples, the number of detected Demodex mites by fluorescence staining was also significantly higher ([11.4 ± 4.2]/cm 2) than that by SSSB ([9.8 ± 4.8]/cm 2; t = 4.77, P < 0.001) . Conclusion:Compared with direct microscopic examination and SSSB, fluorescence staining was more sensitive in the detection of Demodex mites, with better consistency between different observers and shorter time for reading slides.