Purpose <p>Precise tumor excision is important but challenging in breast-conserving surgery (BCS). Tumor-specific fluorescence imaging may be used for intraoperative tumor detection and, therefore, to guide precise tumor excision. The aims of this study are to develop a glucose transporter 1 (GLUT1)-targeted near-infrared fluorescence tracer and evaluate its accuracy for breast cancer detection using fresh surgical breast specimens.</p> Methods <p>Bioinformatic analysis was performed to compare GLUT1 expression between breast cancer and normal breast tissues. A GLUT1-targeted fluorescence imaging tracer WZB117-CY7.5 was developed. In combination with fluorescence imaging (FMI), its binding specificity to GLUT1 was examined in in vitro breast cancer cell experiments, in vivo 4T1 breast tumor-bearing mouse models, and 60 freshly resected human breast tumor tissues. The diagnostic accuracy of WZB117-CY7.5, was evaluated in fresh specimens derived from 60 patients diagnosed with breast cancer.</p> Results <p>GLUT1 expression is higher in breast cancer tissues compared with normal tissues. WZB117-CY7.5 specifically bound to breast cancer cells in in vitro cell experiments and accumulated in tumor areas in a 4T1 tumor-bearing mice after intravenous injection by FMI. Moreover, WZB117-CY7.5 specifically bound to freshly resected human breast cancer and demonstrated excellent diagnostic performance in discriminating breast cancer, irrespective of cancer subtype, from normal breast tissue on fresh surgically resected breast tissues.</p> Conclusions <p>WZB117-CY7.5 showed high accuracy in intraoperative breast cancer detection, irrespective of the cancer subtype. This highlights its potential for clinical applications as a generic tracer for fluorescence image-guided surgery (FIGS) in BCS and fluorescence image-guided pathology for tissue sampling.</p>