Objective:To observe the distribution characteristics of fungi, bacteria and Demodex in the eyelid margin of patients with blepharitis and without blepharitis at different ages. Methods:A cross-sectional study was conducted.A total of 98 patients diagnosed with anterior blepharitis and 99 patients diagnosed with posterior blepharitis in Henan Eye Hospital from March 2021 to June 2022 were enrolled as anterior blepharitis group and posterior blepharitis, respectively.Additionally, 100 patients with an initial diagnosis of refractive error and 200 patients with vitreous opacity were enrolled during the same period as a non-blepharitis group.All patients underwent examinations for lid margin fungi, bacteria and eyelash Demodex, as well as fungal spores and ciliary Demodex count.The differences in the positive rate and load of palpebral fungi, bacteria and eyelash Demodex were compared between anterior and posterior blepharitis groups, as well as across different ages in non-blepharitis group.This study protocol was approved by the Ethics Committee of Henan Eye Hospital (No.HNEECKY-2019[18]).All patients were informed about the purpose and methods of the study.Written informed consent was obtained from each patient. Results:There were significant differences in the positive rates of bacteria, fungi and Demodex and the load of Demodex in the non-blepharitis group at different ages ( χ2=28.34, 10.36, 51.57, H=35.66; all at P<0.01).The positive rates of palpebral bacteria and ciliary Demodex and the load of Demodex were significantly higher and the palpebral fungi positive rate was significantly lower in the ≥60 years old than in the <60 years old (all at P<0.05).There were significant differences in the positive rates of bacteria and fungi among anterior blepharitis, posterior blepharitis and non-blepharitis groups ( χ2=18.99, 6.36; all at P<0.01).The palpebral bacteria positive rate was significantly higher in anterior blepharitis group than in posterior blepharitis and non-blepharitis groups, and the palpebral fungi positive rate was significantly higher in anterior blepharitis and posterior blepharitis groups than in non-blepharitis group (all at P<0.05).There was no significant difference in the ciliary Demodex detection rate among the three groups ( χ2=0.16, P=0.74).The number of palpebral fungi spores and eyelash Demodex counts were higher in anterior and posterior blepharitis groups than in non-blepharitis group, and the differences were statistically significant (all at P<0.05).The positive rate of palpebral margin bacteria in ciliary Demodex-positive group was 45.7%(156/341), which was significantly higher than 25.6%(40/156) in ciliary Demodex-negative group ( χ2=17.20, P<0.01), and there was no significant difference in the positive rate of palpebral margin fungi between them ( χ2=0.11, P=0.70). Conclusions:In the population with normal eyelid margin, the infection of Demodex and bacteria in lid margin increases and fungal infection decreases in the ≥60 years old.Fungal and bacterial infections are the main sources of palpebral infection in patients with blepharitis, and positive detection of Demodex increases the chance of bacterial infection.