Background Current clinical tests for middle ear (ME) injuries and related conductive hearing loss (CHL) are lengthy and costly, lacking the ability to noninvasively evaluate both structure and function in real time. Optical coherence tomography (OCT) provides both, but its application to the audiological clinic is currently limited. Objective Adapt and use a commercial Spectral-Domain OCT (SD-OCT) to evaluate anatomy and sound-evoked vibrations of the tympanic membrane (TM) and ossicles in the human ME. Materials and Methods SD-OCT was used to capture high-resolution three-dimensional (3D) ME images and measure sound-induced vibrations of the TM and ossicles in fresh human temporal bones. Results The 3D images provided thickness maps of the TM. The system was, with some software adaptations, also capable of phase-sensitive vibrometry. Measurements revealed several modes of TM vibration that became more complex with frequency. Vibrations were also measured from the incus, through the TM. This quantified ME sound transmission, which is the essential measure to assess CHL. Conclusion and Significance We adapted a commercial SD-OCT to visualize the anatomy and function of the human ME. OCT has the potential to revolutionize point-of-care assessment of ME disruptions that lead to CHL which are otherwise indistinguishable via otoscopy.