AIM: To develop a novel approach called the Autoacuity Tester, and to evaluate its validity, especially the sensitivity and specificity for detecting amblyopia. METHODS: Children aged from 3 to 12y (n=552) were enrolled in the study. The validity of the Autoacuity Tester was evaluated by comparing it to the Tumbling E Early Treatment Diabetic Retinopathy Study (ETDRS) acuity chart for school age children, and Lea Symbols and Teller acuity card (TAC) for preschool children. The repeatability was assessed by coefficient of repeatability (COR). The sensitivity and specificity for detecting amblyopia were calculated. RESULTS: The mean difference (95% limits of agreement) between the Autoacuity Tester and the ETDRS tests were -0.03 (-0.24, 0.19) logMAR for the school age group. In preschool children, the mean difference was 0.04 (-0.14, 0.21) logMAR between the Autoacuity Tester and the TAC and 0.00 (-0.17, 0.18) logMAR between the Autoacuity Tester and the Lea Symbols. For the school age group, the COR was 0.20 logMAR for the Autoacuity Tester and 0.18 logMAR for the ETDRS. For the preschool group, the COR was 0.13 logMAR for the Autoacuity Tester and 0.21 logMAR for TAC. The Autoacuity Tester (88%) is more sensitive than TAC (72%) in detecting amblyopia (P=0.04), while had similar specificity (92% vs 90%, P=0.20). CONCLUSION: The Autoacuity Tester provides a reliable alternative for assessing visual acuity, and offers advantage of higher testability and repeatability for preschool children.