An ideal 3D cartilage framework and accurate anatomical location are the most important factors to carry out a satisfactory reconstruction of the ear. To streamline this process, we developed an augmented reality assistance system, HoloLens Ear Image Guidance, which is based on computed tomography (CT) data, tailor-made navigation kits to guide reconstruction, data processing software, and HoloLens hardware. The objective of this study is to verify its feasibility in a clinical setting.This study first validated our system in healthy controls and then extrapolated data to test on patients with microtia. First, three healthy volunteers were recruited, and reconstructive navigation kits were made using 3D printing. CT data were collected for the head and neck and imported into the HoloLens Ear Image Guidance Application to generate a personalized 3D virtual ear image. Volunteers then wore the navigation kits while researchers observed them through the HoloLens to check accuracy, track delay, and view the ear image guide.Ten patients with unilateral microtia were recruited and CT data were collected, and reconstructive navigation kits were made to assist with surgery. The procedure was monitored to record the surgeon's experience wearing the HoloLens, the patients' complications associated with wearing navigation kit, and to measure the symmetry between the reconstructed ear and the reference counterpart.In control patients, the deviation between the virtual image and the real ear was less than 2.4% (±0.22%); the tracking delay was less than 1.26 s (±0.09 s), the display effect was good, and surgeons did not report discomfort or dizziness while wearing the HoloLens. Volunteers did not report any pain from holding the navigation reference in their mouth during the test. Following validation, the HoloLens-assisted procedures were not associated with surgeon discomfort or dizziness. No complications were noted in patients including injury to the oral mucosa. Symmetry between the reconstructed ear and the contralateral ear was noted to be satisfactory in HoloLens-assisted surgery.The HoloLens Ear Image Guidance initially met clinical demands in registration accuracy, tracking speed, and subjective user experience, which can be used as the basis for continual software improvements and clinical application.Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.