OBJECTIVE The aim of this study was to evaluate the middle and inner ear function and hearing status of Ankylosing spondylitis (AS) patients. METHODS One hundred twenty-four ears of 62 patients with AS and 90 ears (control group) of 45 healthy subjects were included in the study. The hearing levels of the participants were assessed with pure tone and high-frequency audiometry at the octave frequency between 250 and 16,000 Hz. The absorbance rates and resonance frequencies of middle ear were measured with the wideband tympanometry (WBT) test. AS group was divided into subgroups based on the disease activity, duration of follow-up, medications used for AS, and the subgroups were compared according to hearing status and absorbance and resonance frequencies of middle ears. RESULTS A statistically significant difference was found between the AS and control groups in terms of air and bone conduction thresholds at frequencies of 250, 500, 1000, 2000, and 4000 Hz and the mean PTA1, PTA2, and PTA3 values (p < 0.05). In contrast, no statistically significant difference was observed between two groups in terms of high-frequency thresholds (8000-16,000 Hz). Although the middle ear resonance frequency obtained from the WBT test was higher in the AS group compared to the control group, no significant difference was observed (p > 0.05). The severity of disease adversely affected the hearing threshold at 250, and 500 Hz for air conduction, at 500 Hz for bone conduction threshold, and at PTA1 (p < 0.05). The duration and severity of disease did not affect absorbance values of WBT (p > 0.05). CONCLUSIONS To our knowledge, this is the first study to demonstrate the effects of AS patients on the middle ear function with WBT and to report middle ear absorbance values and resonance frequency changes in AS patients. The higher resonance frequency values found by WBT in AS patients may be due to the stiffness that develops as a result of middle ear involvement. According to pure tone and high-frequency audiometry findings, it has been seen that AS leads to SNHL especially at low frequencies.