To investigate the prevalence and influencing factors of Demodex folliculorum (DF) in the eyelashes of healthy young males. An epidemiological cross-sectional prevalence study was conducted. We conducted visual acuity, eye-related examination, eyelash microscopic examination and DF count of recruits in Fujian Province in 2019. The presence of DF was analyzed according to age, keratorefractive surgery, annual household income, educational level, long-term residence, sleep time, time of using electronic products, smoking and drinking habit. A total of 2253 healthy young males (aged 17–24 years) were studied for the presence of DF within eyelash follicles. The total prevalence of DF was 20.73% in our study. Subjects with history of keratorefractive surgery had a statistically significant increase in the prevalence of DF (P < 0.001). The prevalence of DF was higher in subjects with ocular symptoms than in those without symptoms (P < 0.05). Factors such as the educational level and so on we analyzed had no significant correlation with the prevalence of DF (all P > 0.05). According to the multivariate logistic regression analysis, the history of keratorefractive surgery was the risk factors of DF infection (P < 0.001), and the risk of infection was 1.437 times higher in the population with the history of keratorefractive surgery than in the population without. There was no correlation between ocular discomfort and DF infection (P > 0.05). The prevalence of DF in eyelash follicles in healthy young males was relatively high. The history of keratorefractive surgery was an important risk factor for the infection.