Objectives <p>To analyze the relationship between laryngopharyngeal reflux (LPR) represented by pepsin and trypsin, and pathogenesis of otitis media with effusion (OME) in children with adenoid hypertrophy.</p> Methods <p>Children with OME who required adenoidectomy and tympanostomy/tympanostomy tubes placement were enrolled in OME group, whereas children with adenoid hypertrophy (AH) who only required adenoidectomy were enrolled in AH group, respectively. Pepsin and trypsin concentrations were evaluated by enzyme-linked immunosorbent assay in adenoid tissue from the OME and AH groups. Pepsin and trypsin concentrations were also evaluated by enzyme-linked immunosorbent assay in middle ear effusion (MEE) and plasma from the OME group.</p> Results <p>The size of adenoids and the concentrations of pepsin and trypsin in the adenoid tissue between the OME group and the AH group had no statistically significant differences. The concentrations of pepsin and trypsin in MEE were 157.30 ± 182.59ng/mL and 1144.79 ± 1302.85ng/mL, respectively, which were significantly higher than those in the serum (<i>P</i> &lt;.05). Furthermore, the concentration of trypsin in MEE was significantly higher than that of pepsin (<i>P</i> &lt;.05).</p> Conclusions <p>The concentrations of pepsin and trypsin in adenoids cannot be used as separate evaluation indicators for whether OME will be concurrented. In addition, pepsin and trypsin in MEE are probably caused by LPR and may be involved in the pathogenesis of OME. And, trypsin may have a greater destructive effect on the middle ear cavity.</p>