Objective To investigate the presence of pepsin and trypsin in the middle ear effusion of children with acute suppurative otitis media(ASOM).Methods Middle ear effusion samples were collected from 71 children with ASOM at Children's Hospital of Xuzhou.According to the characteristics of the middle ear effusions,the effu-sion was divided into serous and mucous types.The pH testing,Western Blotting(WB),and enzyme-linked immu-nosorbent assay(ELISA)were performed.Results ① There were 49.29%(35/71)of ASOM patients had a posi-tive RSI score(>13).② The positive rate of pepsin in ASOM children was 49.29%(35/71),and the positive rate of trypsin was 42.25%(30/71).In addition,the positive rate of pepsin in RSI-positive children was 100%(35/35),and the positive rate of trypsin was 60%(21/35).There was no significant difference in the positive rate of pepsin and trypsin between serous and mucous middle ear effusion(P>0.05).③ The pepsin concentration was 47.80(39.80,69.30)ng/ml and the trypsin concentration was 291.87±20.45 ng/ml in middle ear effusion of chil-dren with ASOM who had a positive WB test,and the trypsin concentration was significantly higher than pepsin(P<0.05).There was no significant difference between the pepsin and the trypsin concentrations in serous and mu-cous middle ear effusion(P>0.05).④ The pH value of mucous middle ear effusion was 7.39±0.28,and the pH value of serous middle ear effusion was 7.36±0.26.There was no significant difference between the pH value in se-rous and mucous middle ear effusion(P>0.05).Conclusion The detection rates of pepsin and trypsin in middle ear effusion of children with ASOM were high which has important diagnostic value for children with ASOM combined with LPRD.