To explore the accuracy of VR technology for strabismus screening.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 33 cm and 6 m. 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.A total of 88 healthy individuals, 81 exotropia patients, 17 esotropia patients, and five 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 < .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 < .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 < .001). For ocular deviation angle calculations, intraclass correlation coefficient (ICC) was used to compare the agreement between VR and manual results. It showed moderately agreement for exotropia in the near mode (ICC = 0.587, range, -0.036-0.826) and less agreement for exotropia in far mode (ICC = 0.349, range, -0.042-0.615). ICC was in good agreement (ICC = 0.701, range, 0.358-0.879) for the esotropic far mode results (6 m) and not statistically significant for the esotropic near mode results (33 cm). ICC was in good agreement (ICC = 0.606, range = -0.143 to 0.946) for the vertical-near-mode results (33 cm) and not statistically significant for the vertical-far-mode results (6 m).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.© The Author(s) 2024.