We aimed to study middle ear barotrauma caused by fast compression followed by buoyant ascent escape from 200 m underwater and its effect on the auditory system, and to validate the preventive effect of tympanocentesis on middle ear barotrauma. Twenty Sprague Dawley rats were divided into two groups: rats in group A underwent a simulated fast buoyant ascent escape from a depth of 200 m, while those in group B underwent tympanocentesis before the procedure described for group A. Ear endoscopy, acoustic conductance, and auditory brainstem response (ABR) tests were conducted before and after the procedure to evaluate the severity of middle ear barotrauma and auditory function in both groups. Additionally, histopathological examination of the middle ear in both groups was conducted to evaluate the severity of middle ear barotrauma by observing submucosal haemorrhage. None of the ears in either group showed any abnormalities before the experiment. In group A, middle ear barotrauma was universally observed after the simulation procedure. The tympanograms of all ears were initially type A and became type B after the procedure. Further, after the simulation, the hearing thresholds at different frequencies (4, 8, 16, 24, and 32 kHz) assessed by ABR significantly increased compared to those before the procedure. In group B, no middle ear barotrauma was observed, and the hearing threshold at each frequency did not change significantly compared with post-puncturing. After dissecting the middle ear, gross pathological observations were consistent with the above results. Microscopically, blood accumulation and submucosal haemorrhage in the middle ear cavity were observed in group A but not in group B. Fast buoyant ascent from 200 m underwater can cause middle ear barotrauma, resulting in hearing loss. Tympanic membrane puncture can effectively prevent middle ear barotrauma caused by the rapid buoyant ascent escape procedure.