PURPOSE: To report the capability of autofluorescence as a novel approach to diagnosing macular holes. DESIGN: Observational case report. METHODS: In an institutional study, autofluorescence photos and optical coherence tomography scans of two patients who had surgery for macular holes were reviewed. RESULTS: Macular hole cases had a strong subfoveal autofluorescence signal, which disappeared after surgical closure of the macular hole. CONCLUSIONS: Autofluorescence is a novel approach to demonstrating a macular hole. This technique can also be used to confirm postoperative outcome after macular hole repair. A lack of autofluorescence corresponds to macular hole closure.