Despite the multitude of therapeutic agents available to treat prostate cancer (PC), there are still no effective and safe measures to treat the tumor. It remains a challenge to develop a simple approach to target PC with specific antibodies. In our study, D2B monoclonal antibodies against a prostate-specific membrane antigen (PSMA) were used. We investigated the functionalization of gold nanoparticles (AuNPs) with D2B to generate favorable physicochemical and biological properties that mediate specific binding to PC. For this purpose, AuNPs with a size of about 25 nm were synthesized in water using sodium citrate as a reducing and stabilizing agent and then coated with D2B. Major physicochemical properties of naked and D2B-coated AuNPs were investigated by ultraviolet–visible (UV–vis) spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. The successful binding of D2B to AuNPs-citrate caused a 15 nm red shift in the UV–vis. This was assessed by DLS as an increase in zeta potential from ∼−45 to ∼−23 mV and in the size of AuNPs from ∼25 to ∼63 nm. Scanning electron microscopy confirmed the size shift of AuNPs, which was detected as an exterior organic layer of D2Bs surrounding each AuNP. Even at high exposure levels of the bioconjugates, PSMA-PC-3 cells exhibited minimal cytotoxicity. The specific and dose-dependent binding of AuNPs-D2B to PC-3-PSMA cells was validated by flow cytometry analysis. Our data provide effective drug delivery systems in PC theranostics.