Objective To investigate the changes of nitric oxide (NO) and inflammatory factors in the ear effusion in patients with secretory otitis media. Methods Ninety patients with secretory otitis media admitted to Joint Support Force 908 Hospital from January 2022 to April 2023 were selected as an observation group, and 90 healthy individuals during the same period were selected as a control group. In the observation group, there were 54 males and 36 females, aged (39.18±3.19) years, 58 cases in mucous stage and 32 cases in serous stage, 38 cases in acute stage and 52 cases in chronic stage. In the control group, there were 50 males and 40 females, aged (40.53±4.21) years. The levels of NO, γ-interferon (IFN-γ), and interleukin-2 (IL-2) in the ear effusion and levels of immunoglobulin (Ig) and peripheral blood T lymphocyte subsets were compared. Independent sample t test and χ2 test were used for statistical analysis. Results The levels of IgM, IgG, and IgA in the observation group were higher than those in the control group (all P<0.001). The levels of CD4/CD8, CD4+, and CD3+ in peripheral blood T lymphocyte subsets were lower than those in the control group, and the level of CD8+ was higher than that in the control group (all P<0.001). The levels of IL-2, IFN-γ, NO, IgM, IgG, IgA, CD4/CD8, CD8+, CD4+, and CD3+ in the acute stage patients were (10.79±1.43) mg/L, (75.42±18.67) µg/L, (2.69±0.49) mg/L, (3.81±0.36) g/L, (19.92±2.28) g/L, (3.81±0.33) g/L, (1.59±0.24), (23.40±3.42)%, (42.31±4.23)%, and (61.38±5.17)%, those in the chronic stage patients were (13.35±1.48) mg/L, (108.72±21.34) µg/L, (4.38±0.58) mg/L, (5.41±0.38) g/L, (33.46±2.53) g/L, (4.96±0.41) g/L, (1.32±0.15), (34.23±3.62)%, (38.11±0.04)%, and (55.30±4.94)%, with statistically significant differences (t=8.221, 7.701, 14.557, 20.169, 26.130, 14.239, 6.554, 14.346, 7.175, and 5.655; all P<0.001). The levels of IL-2, IFN-γ, NO, IgM, IgG, IgA, CD4/CD8, CD8+, CD4+, and CD3+ in the mucous stage patients were (15.62±1.65) mg/L, (111.73±22.14) µg/L, (5.08±0.63) mg/L, (34.18±2.49) g/L, (5.13±0.42) g/L, (1.34±0.18) g/L, (1.30±0.14), (35.61±3.59)%, (37.52±4.12)%, and (56.48±4.52)%, those in the serous stage patients were (9.90±1.39) mg/L, (70.42±17.80) µg/L, (2.67±0.52) mg/L, (3.87±0.32) g/L, (20.41±2.35) g/L, (3.93±0.34) g/L, (1.72±0.23), (25.63±3.54)%, (41.92±4.24)%, (60.82±5.24)%, with statistically significant differences (t=16.615, 9.056, 18.438, 68.378, 48.349, 47.347, 10.775, 12.686, 4.800, and 4.118; all P<0.001). Conclusion For patients with secretory otitis media, the degree of disease can be comprehensively understood by the indicators of immune status and IL-2, IFN-γ, and NO in the ear effusion, with high clinical application value.