Background: Tympanosclerosis is the calcification of middle ear connective tissue, including tympanic membrane, which sometimes occurs after middle ear inflammation. Tumor necrosis factor (TNF)-α tends to induce and maintain inflammatory responses in various diseases, including chronic otitis media with effusion (COME). Aim: To assay plasma level of TNF-α gene expression in patients with tympanosclerosis due to ventilation tube insertion. Methods: Sixty patients were included in this study. Patients were divided into two groups: Group A: 60 patients, according to inclusion and exclusion criteria, and Group B: 20 volunteers. Patients were subjected to history taking, full clinical examination, and laboratory investigation of TNF-α gene expression [quantitative real-time PCR (qRT-PCR)]. Results: There was a statistical difference between groups according to TNF-α gene expression, as the mean TNF-α in the case group (3.87) was statistically higher than in the control group I, p < 0.001. There was a statistical difference in TNF-α level regarding the number of tube insertion (p= 0.005) and duration of secretory otitis media (OM) (p= 0.042). However, there was no statistical difference in TNF-α level regarding medication use or episodes of acute OM. Conclusion: The degree of gene profile expression for TNF-α in selected middle ear structures with tympanosclerosis was statistically important, higher than the control group