Precise identification of the tumor margins during breast-conserving surgery (BCS) remains a challenge given the lack of visual discrepancy between malignant and surrounding normal tissues. Therefore, we developed a fluorescent imaging agent, ICG-p28, for intraoperative imaging guidance to better aid surgeons in achieving negative margins in BCS. Here, we determined the pharmacokinetics (PK), biodistribution, and preclinical toxicity of ICG-p28. The PK and biodistribution of ICG-p28 indicated rapid tissue uptake and localization at tumor lesions. There were no dose-related effect and no significant toxicity in any of the breast cancer and normal cell lines tested. Furthermore, ICG-p28 was evaluated in clinically relevant settings with transgenic mice that spontaneously developed invasive mammary tumors. Intraoperative imaging with ICG-p28 showed a significant reduction in the tumor recurrence rate. This simple, nontoxic, and cost-effective method can offer a new approach that enables surgeons to intraoperatively identify tumor margins and potentially improves overall outcomes by reducing recurrence rates.