Objective To investigate the correlation between eyelash Demodex infection and meibomian gland dysfunction (MGD) and to analyze the effect of eyelash Demodex infection on ocular surface symptoms and signs in patients with MGD. Methods Prospective cohort study. 47 patients with MGD diagnosed in the ophthalmology clinic of Guangzhou First People's Hospital from July 2018 to March 2019 were selected. The MGD patients were assigned to the MGD＜40 years old group (A₁ group, n=28) and MGD＞60 years old Group (B₁ group, n=19) according to the age. According to the Demodex test results, MGD patients were assigned to the Demodex-positive group (I group, n=22) and the Demodex-negative group (II group, n=25). At the same time, 49 patients without MGD or only with ametropia and vitreous opacity were selected and assigned to the NMGD＜40 years old group (A₂ group, n=30) and NMGD＞60 years old group (B₂ group, n=19) according to their age. The incidence of ocular symptoms (itchiness, pain, dryness, foreign body perception, and blurred vision), the number and rate of detection of eyelash Demodex, meibomian gland ejection score, meibomian gland secretion score, and Ocular Surface Disease Index (OSDI) were analyzed. Results The number of eyelash Demodex was (1.14±1.938) in MGD＜40 years old group (A₁ group) and (0.17±0.531) in NMGD＜40 years old group (A₂ group). The difference in the number of eyelash Demodex between these two groups was found to be statistically significant (P=0.003,＜0.05). The positive rate of eyelash Demodex was 43 % in MGD＜40 years old group (A1 group) and 10% in NMGD＜40 years old group (A₂ group). The difference in the positive rate of eyelash Demodex between these two groups was found to be statistically significant (P=0.006,＜0.05). The number of eyelash Demodex in MGD＞60 years old group (B₁ group) was (1.89±2.331) and in NMGD＞60 years old group (B₂ group) was (0.68±1.529). The difference in the number of eyelash Demodex between these two groups was not statistically significant(P>0.05). The positive rate of eyelash Demodex was 53 % in MGD＞60 years old group (A₁ group) and 21% in NMGD＞60 years old group (A₂ group). The difference in the positive rate of eyelash Demodex between these two groups was not statistically significant (P>0.05). The score of the meibomian gland secretion in the MGD with eyelash Demodex group (I Group) was (3.00±1.11) and in the MGD without eyelash Demodex group (II group) was (2.28±1.28). The difference in the score of the meibomian gland secretion between these two groups was found to be statistically significant (P=0.047<0.05). The differences of the OSDI score, the meibomian gland discharge ability score, the incidence rate of dry eye, eye pain, eye itching, foreign body sensation and blurred vision between these two groups were not statistically significant (P>0.05). The difference in the culture level between the MGD group and the NMGD group was not statistically significant (P=0.382, >0.05). Conclusion First, in the population under 40 years old, eyelash Demodex is correlated with MGD. Then in the population over 60 years old, the effect of eyelash Demodex on the formation of MGD compared with other influencing factors is not significant. The results indicated that the effect of eyelash Demodex was mainly reflected in the middle and young age, and the correlation between them was further reduced with the increase of age. In addition, in MGD patients, eyelash Demodex infection was not significantly correlated with the incidence of eye pain, eye dryness, eye itching, foreign body sensation, and blurred vision, as well as the degree of impairment of meibomian gland secretion function.