Abstract: OBJECTIVE To explore the expression levels of serum inflammatory factors and proteins and messenger-ribonucleic acid（mRNA） of Toll-like receptor 2（TLR2） and TLR4 in middle ear secretions of patients with fungal otitis media. METHODS A total of 102 patients with fungal otitis media who were treated from Dec 2018 to Dec 2019 were assigned as the case group, and 40 otosclerosis patients without external ear canal infection were chosen as the control group. The levels of serum interleukin-6（IL-6）, tumor necrosis factor-α（TNF-α） and macrophage inflammatory protein-2（MIP-2） were detected, the middle ear granulation tissue specimens were collected from the patients of the case group during surgery, and the middle ear mucosa specimens were collected from the patients of the control group. The expression levels of TLR2 and TLR4 were detected by immunohistochemical staining, and the mRNA levels of TLR2 and TLR4 were detected by means of reverse transcription polymerase chain reaction（RT-PCR）. RESULTS Totally 102 strains of fungi were isolated, 35（34.32%） of which were Aspergillus, 28（27.45%） were Penicillium, and 23（22.55%） were Candida albicans. The levels of serum IL-6, TNF-α and MIP-2 of the case group were significantly higher than those of the control group（P＜0.05）.The expression levels of proteins and mRNA of TLR2 and TLR4 were significantly higher in the middle ear granulation tissue specimens of the case group than in the middle ear mucosa specimens of the control group（P＜0.05）. CONCLUSION The molecular mechanism of fungal otitis media may be related to the up-regulated expression of TLR2 and TLR4 genes.