Background Vision-threatening diabetic retinopathy (VTDR) is a microvascular retinal complication caused by diabetes mellitus, which may lead to blindness if left untreated. One of the most effective methods to prevent diabetic-related ocular complications is through diabetic retinopathy (DR) screening. The community rarely carries out diabetic retinopathy-related eye examinations because using non-portable fundus photographs as its gold standard is costly and impracticable. This study aimed to determine the accuracy of smartphone-based fundus photographs as a practical and affordable tool for VTDR screening in developing countries. Methods This cross-sectional study used a consecutive technique at Cicendo National Eye Hospital, Indonesia. Patients with diabetes mellitus aged ≥20 years were evaluated for two-field mydriatic fundus photos using a non-portable fundus photo and a smartphone- based fundus photo utilizing the i-Spot fundus adapter. Results were analyzed to determine diagnostic test parameters. Results Two hundred and nineteen two-field mydriatic fundus photos were obtained from 139 patients. Smartphone-based fundus photography demonstrated a sensitivity of 98.4% (CI 96.6–100%), a specificity of 87.1% (CI 75.3–98.9%), a positive predictive value of 97.9% (CI 95.9–99.9%), a negative predictive value of 90.0% (CI 79.3–100%), and an accuracy of 96.8% (CI 94.5–99.8%). Conclusion The use of smartphone-captured fundus images proves to be a reliable screening method for VTDR. This tool has the potential to effectively screen the population, helping prevent future visual loss attributed to the disease.