Multimodal biosafety assessments indicated favorable biocompatibility with no evident acute toxicity and low immunogenic potential.<h4>Conclusions</h4>PSMA-12-IRDye800CW enables NIR-II fluorescence imaging-assisted intraoperative navigation and provides a quantitative, pathology-anchored readout for histopathological margin mapping in prostate cancer, supporting further clinical validation of this PSMA-targeted strategy for fluorescence-guided surgery and margin assessment. This evidence concerns the gene FOLH1 and prostate carcinoma.