Here, we developed a PSMA-targeted near-infrared fluorescent probe, PSMA-12-IRDye800CW, that leverages the clinically used IRDye800CW scaffold and its extended emission tail beyond 1000 nm to support NIR-II fluorescence imaging for intraoperative navigation and histopathological margin mapping.<h4>Methods</h4>PSMA-12-IRDye800CW integrates a PSMA-targeting ligand with an albumin-binding linker to enable active targeting and circulation-assisted tumor accumulation. The gene discussed is FOLH1; the disease is neoplasm.