Background To explore the accuracy and reproducibility of VR technology for strabismus screening.Methods In this cross-sectional study, a total of 191 subjects were included. Examinations included VR technology based on infrared, the cover-uncover test, and the prism alternating cover test (PACT) at various gaze positions, specifically at distances of 33cm and 6m. The accuracy of VR in diagnosing strabismus and quantifying the degree of strabismus was evaluated by comparing the VR results to the average outcomes of manual examinations conducted by the two physicians, considered the gold standard.Results A total of 88 healthy individuals, 81 exotropia patients, 17 esotropia patients, and 5 vertical strabismus patients were included in this study, and each subject was screened for less than 2 min. The sensitivity of VR for screening strabismus was 80.3% and the specificity was 80%, displaying moderately consistent with the manual results (Kappa = 0.598, p < 0.001). The sensitivity for diagnosing exotropia was 76.5% and the specificity was 91.8%, also showing strong agreement with the manual results (Kappa = 0.695, p < 0.001). The sensitivity for diagnosing esotropia was 94.1% and the specificity was 95.4%, with strong agreement with the manual results (Kappa = 0.755, p < 0.001). The sensitivity for diagnosing vertical strabismus was 80% and the specificity was 98.9%, with strong agreement with manual results (Kappa = 0.719, p < 0.001). Reproducibility of VR compared to manual results, expressed as intraclass correlation efficient (ICC), was moderately reproducible for exotropia in near mode (ICC = 0.587, range, -0.036-0.826) and less reproducible for exotropia in far mode (ICC = 0.349, range, -0.042-0.615). ICC was highly reproducible (ICC = 0.701, range,0.358–0.879) for the esotropic far mode results (6m) and not statistically significant for the esotropic near mode results (33cm). ICC was moderately reproducible (ICC = 0.606, range, -0.143-0.946) for the vertical-near-mode results (33cm) and not statistically significant for the vertical-far-mode results (6m).Conclusions This novel technique for strabismus screening, which utilizes virtual reality technology, enables quick, precise, and straightforward diagnosis of strabismus. It has also shown notable efficacy in accurately measuring the angle of deviation in cases of exotropia.