Objective. In this study, we aimed to identify mt3243A > G mutation carriers in a group of Chinese elderly type 2 diabetic patients by a rapid and noninvasive diagnostic system. Methods. DNA was extracted from blood, saliva, and urine sediment samples. The mutation screening and quantitation of heteroplasmy were performed by high-resolution melting (HRM) curve and pyrosequencing, respectively. Patients with mt3243A > G mutation underwent a detailed audiometric, ophthalmologic, neurological, and cardiac examination. Results. Two patients (2/1041) carrying the mt3243A > G mutation were detected among all type 2 diabetic patients. In patient 1, the heteroplasmy was 0.8%, 2.8%, and 14.7% in peripheral blood leukocytes, saliva, and urine sediment, respectively. In patient 2, the heteroplasmy was 5.3%, 8.4%, and 37.7% in peripheral blood leukocytes, saliva, and urine sediment, respectively. Both of the two patients showed hearing impairment. Abnormal ophthalmologic conditions and hyperintensity on T2-weighted magnetic resonance images were showed in patient 1. Conclusion. The occurrence of mt3243 A > G mutation was 0.2% in Chinese elderly type 2 diabetic patients. Moreover, detection of mt3243 A > G mutation in urine sediment with high-resolution melting (HRM) curve and pyrosequencing is feasible in molecular genetic diagnosis.