Background: To obtain high-yield histological samples by targeted prostate cancer (PCa) biopsy is the current trend compared with transrectal ultrasound (TRUS)-guided systematic histological biopsy, which is regarded as the gold standard for prostate cancer (PCa) diagnosis. In this paper, we present a targeted PCa imaging strategy using a real-time molecular photoacoustic imaging system integrated with a handheld US probe (PAI/US) and synthesized an integrin α v β 3 targeted probe based on ICG (cRGD–ICG). Results: A small molecular PAI probe was synthesized and exhibited excellent targeted imaging ability in vitro . In vivo photoacoustic imaging was carried out after intravenous injection of cRGD-ICG in orthotopic PCa-bearing rats under the facilitation of the PAI/US system. Maximum molecular photoacoustic signals were observed in the tumor area in vivo after the probe injection, which showed 3.8-fold higher signal enhancement than that in the control group ( P < 0.05). Significantly higher cRGD-ICG accumulation was observed under confocal microscopy in the tumor region than in normal prostate tissue. Conclusions: All our results showed that the comprehensive strategy provided a high-yield and reliable method for PCa diagnosis and targeted prostate biopsy, with great clinical translation potential.