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Background: Blood-injection phobia is a chronic and debilitating disorder in children and adolescents which can be associated with anxiety avoidance of many essential treatments. Although there are some current recommended treatments for blood-injection phobia, little evidence is available on the virtual reality therapy. Therefore, in this study we aimed to investigate the effect of virtual reality therapy on blood-injection phobia among children and adolescents. Methods: In this quasi-experimental study, 31 children and adolescents aged 6 to 18 years with the diagnosis of blood-injection phobia were investigated. All children received 12 sessions of working with virtual reality software. The children's anxiety level was assessed using Spielberger and specific symptoms of blood-injection phobia questionnaires. Statistical analysis was performed using SPSS software. Findings: After working with the software, the mean scores of state, trait, and state-trait anxiety and also blood-injection phobia in children were significantly lower than their levels before working with the software (P < 0.050 for all). There was a direct and significant correlation between age and the degree of reduction of situational anxiety (r = +0.60, P < 0.001). Conclusion: Virtual reality therapy can effectively reduce state, trait, and state-trait anxiety and also blood-injection phobia in children and adolescents. However, further studies are required to determine the best version of virtual reality software for blood-injection phobia.