Development of specific molecular probes holds great promise for early diagnosis of aggressive prostate cancer (PCa). Here, 2-[3-(1,3-dicarboxypropyl) ureido] pentanedioic acid (DUPA)-conjugated ligand and bis-isoindigo-based polymer (BTII) are synthesized to formulate semiconducting polymer nanoparticles (BTII-DUPA SPN) as a prostate-specific membrane antigen (PSMA)-targeted probe for PCa imaging in the NIR-II window. Insights into the interaction of the imaging probes with the biological targets from single-cell to whole-organ levels are obtained by transient absorption microscopy (TAM) and photoacoustic tomography (PAT). The targeting mechanism, kinetics, and specificity of BTII-DUPA SPN to PSMA-positive PCa cells are revealed at cellular level with TAM. At the organ level, PAT imaging of BTII-DUPA SPN in the NIR-II window demonstrates superior penetration depth and imaging contrast. By intravenous administration, BTII-DUPA SPN demonstrates selective accumulation and retention in the PSMA-positive tumor, allowing noninvasive PAT detection of PSMA overexpressing PCa. The distribution of nanoparticles at tissue level is further analyzed through TAM. These results collectively demonstrate a new SPN for targeted cancer detection by TAM and PAT imaging.