A wide-field optical coherence tomography (OCT) probe was developed that adapts a diagonal-scanning scheme for three-dimensional (3D) in vivo imaging of the human tympanic membrane. The probe consists of a relay lens to enhance the lateral scanning range up to 7 mm. Motion artifacts that occur with the use of handheld probes were found to be decreased owing to the diagonal-scanning pattern, which crosses the center of the sample to facilitate entire 3D scans. 3D images could be constructed from a small number of two-dimensional OCT images acquired using the diagonal-scanning technique. To demonstrate the usefulness and performance of the developed system with the handheld probe, in vivo tympanic membranes of humans and animals were imaged in real time.