In this paper, we report the successful use of non-cadmium-based Mn-doped ZnSe d-dots (Mn/ZnSe) as highly efficient and nontoxic optical probes for human prostate cancer cells imaging. Mn/ZnSe d-dots are directly prepared in aqueous solution. The α-methylacyl-CoA racemase (AMACR) is overexpressed in prostate cancers; the presence of antibodies specific for AMACR is more sensitive and specific than serum prostate specific antigen levels in distinguishing patients with prostate cancers. Mn/ZnSe d-dots were linked to anti-AMACR to form Mn/ZnSe d-dots-anti-AMACR bioconjugates for the direct prostate cancer cell imaging. 3-(4,5-Dimethylthiazol-2-yl)-2 and 5-diphenyl tetrazolium bromide assay demonstrated that Mn/ZnSe d-dots exhibited favorable cytocompatibility to LNCaP cells with high concentration (1 mM) and long-time incubation (24 h). Furthermore, cellular imaging results demonstrated that Mn/ZnSe d-dots were remarkably efficacious for high-specificity cell imaging. The antibody-mediated delivery of the bioconjugates was further confirmed by the observation of no fluorescence signals in vitro targeting in nonprostate-cancer-based cell lines which are negative for AMACR. Mn/ZnSe d-dots as non-cadmium-based safe and efficient optical imaging nanoprobes could therefore be used for targeting imaging and treatment of cancers in the early stage.