AIM: To investigate the efficacy of a novel screening software for amblyopia. METHODS: Totally 324 preschoolers aged 3-6y old from outpatient department participated in the study in 2017. They were assessed for amblyopia by their parents, using the screening software and were reevaluated by ophthalmologists with professional eye tests. Sensitivity, specificity of the screening software was validated by comparing the results from the screening software and those from the professional tests. RESULTS: All 324 children (176 males and 148 females) completed all the procedures. Four amblyopes were found by professional tests. And 3 of them were detected by the screening software. The sensitivity, specificity was 75.0%, 90.0%, respectively. Professional tests cost an average of US$ 607.5/case of amblyopia detected, whereas the cost was near US$ 0/case for the screening software, except the cost of software development. CONCLUSION: The results from this pilot study indicate that the screening software for amblyopia is a simple, and highly effective, which offers a new option of amblyopia screening for developing countries.