Background and ObjectivesNegative surgical margins are significant in improving patient outcomes. However, surgeons can only rely on visual and tactile information to identify tumor margins intraoperatively. We hypothesized that intraoperative fluorescence imaging with indocyanine green (ICG) could serve as an assistive technology to evaluate surgical margins and guide surgery in bone and soft tissue tumor surgery.MethodsSeventy patients with bone and soft tissue tumors were enrolled in this prospective, non‐randomized, single‐arm feasibility study. All patients received intravenous indocyanine green (0.5 mg/kg) before surgery. Near‐infrared (NIR) imaging was performed on in situ tumors, wounds, and ex vivo specimens.Results60/70 tumors were fluorescent at NIR imaging. The final surgical margins were positive in 2/55 cases, including 1/40 of the sarcomas. Surgical decisions were changed in 19 cases by NIR imaging, and in 7/19 cases final pathology demonstrated margins were improved. Fluorescence analysis showed that the tumor‐to‐background ratio (TBR) of primary malignant tumors was higher than that of benign, borderline, metastatic, and tumors ≥5 cm in size had higher TBR than those <5 cm.ConclusionsICG fluorescence imaging may be a beneficial technique to assist in surgical decision making and improving surgical margins in bone and soft tissue tumor surgery.