Objective To investigate the pathophysiological role of oxidative stress in otitis media with effusion (OME) based on the physical properties of middle ear effusion, and to provide a theoretical basis for the etiology of OME. Methods Clinical data, otoscopic findings, and audiological test results were collected from pediatric OME patients undergoing tympanostomy tube placement in the Department of Otolaryngology-Head and Neck Surgery between July 2023 and December 2024. Concentrations of myeloperoxidase (MPO), catalase (CAT), and malondialdehyde (MDA) were measured in both middle ear effusions (intraoperatively collected) and serum samples (preoperatively collected). Participants were grouped according to effusion viscosity, disease duration, and degree of hearing loss. Differences in MPO, CAT, and MDA levels were compared across groups, as well as between middle ear effusions and matched serum samples. Results A total of 53 pediatric OME patients were included, with a median age of 5.6 (1-13 ) years, including 32 boys and 21 girls. Otoscopic examination confirmed middle ear effusion in all cases. The preoperative average pure-tone air conduction threshold was (36.51±1.31) dB HL. The concentrations of MPO and CAT in the viscous middle ear effusion group were significantly lower than those in the thin middle ear effusion group, while the concentration of MDA was significantly higher than that in the thin middle ear effusion group. The differences were statistically significant (P<0.05). The concentration levels of oxidative stress markers in the middle ear effusion and their own serum of 10 children with OME were compared. It was found that the concentrations of MPO, CAT and MDA in the middle ear effusion were significantly higher than those in their own serum, and the difference was statistically significant (P<0.001). No correlation was found between disease duration or degree of hearing loss and the concentrations of MPO, CAT, or MDA in middle ear effusions (P>0.05). Conclusions The physicochemical properties of middle ear effusion are related to oxidative stress. There are differences in the expression of oxidative stress between middle ear effusion and serum, and oxidative stress may be involved in the pathogenesis of OME.