Abstract Purpose Our purpose was to report the initial clinical experience of intraoperative B-scan ultrasonography in combination with 25-gauge pars plana vitrectomy for severe open globe injury with hemorrhagic retinal and choroidal detachment. Methods Six eyes of six consecutive patients with severe open globe injury underwent intraoperative B-scan ultrasonography and 25-gauge pars plana vitrectomy at Osaka University Hospital in Japan. The feasibility of intraoperative B-scan ultrasonography, best-corrected visual acuity (BCVA), retinal reattachment, and intraoperative and postoperative complications were evaluated. Results Five patients presented with a ruptured globe and one patient with double penetration. Preoperative best-corrected visual acuity was no light perception in four eyes and light perception in two eyes. All patients underwent intraoperative B-scan ultrasonography and 25-gauge pars plana vitrectomy within 12h after open globe injury. Intraoperative B-scan ultrasonography was feasible in all cases and was useful for diagnosing choroidal hemorrhage (four eyes), massive subretinal hemorrhage (two eyes), and retinal detachment (five eyes). In addition, serial real-time B-scan imaging facilitated successful evacuation of the choroidal hemorrhage and massive subretinal hemorrhage by external drainage, resulting in opening of the vitreous space to allow subsequent pars plana vitrectomy without entry site-related complications. After surgery, all patients had successful retinal attachment, and there was no loss of light perception. Conclusion Intraoperative B-scan ultrasonography is technically feasible and may potentially improve the safety and efficacy of severe open globe injury repair.