Selenium-doped nanostructure has been considered as an attractive approach to enhance the antibacterial activity of calcium phosphate (CaP) materials in diverse medical applications. In this study, the selenium-doped biphasic calcium phosphate nanoparticles (SeB-NPs) were first synthesized. Then, silver was in situ incorporated into SeB-NPs to obtain nanostructured composite nanoparticles (AgSeB-NPs). Both SeB-NPs and AgSeB-NPs were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV-Vis), X-ray photoelectron spectroscopy (XPS), and Raman spectra. The results confirmed that the SeO32- was doped at the PO43- position and silver nanoparticles were deposited on the surface of SeB-NPs. Next, Transmission Electron Microscopy (TEM) analysis displayed that the prepared AgSeB-NPs had a needle-cluster-like morphology. CCK-8 analysis revealed SeB-NPs and AgSeB-NPs had good cytocompatibility with osteoblasts. The antibacterial activity of the prepared AgSeB-NPs was confirmed by using Gram-negative E. coli and Gram-positive S. aureus. The above results manifested the significance of the final AgSeB-NPs for biomedical applications.