Otitis media with effusion (OME) is a common middle ear disease characterized by fluid accumulation without acute infection, leading to conductive hearing loss. Conventional diagnostic tools, such as tympanometry and otoscopy, have limited sensitivity and rely on expert interpretation. This study investigates vibro-acoustic radiation (VAR) as a novel, non-invasive, and objective method for OME detection. VAR signals were obtained from 36 OME patients (43 ears) and 15 normal ears using bone-conduction excitation and stereo microphones, and the frequency response functions were analyzed. OME increases the mechanical loading of the tympanic membrane and ossicular chain, thereby modifying sound transmission across the middle ear. Using a simplified theoretical model, we estimated acoustic parameters of the ear canal, eardrum, and middle ear, including specific acoustic impedance and resonance frequency ranges, to interpret changes in VAR. VAR analysis revealed significantly reduced signal amplitude in the 8–10 kHz range in OME ears compared with normal ears (p < 0.05). A classification algorithm based on these features achieved 86.7% accuracy, 85.0% sensitivity, and 80.0% specificity, with an area under the ROC curve of 0.986. These findings suggest that VAR has strong potential as a non-invasive diagnostic tool for OME, warranting validation in larger clinical studies.