Monodispersed self-assembled Eu3+-doped yttrium orthovanadate (YVO4) microspheres have been prepared by a facile hydrothermal method using trisodium citrate (Na3C6H5O7) as surfactants and complexing agents. Then the as-prepared YVO4:Eu3+ microspheres were subjected to the calcination treatment at different temperatures. The phase structure, microstructure, and optical properties of these microspheres were characterized by X-ray diffraction (XRD) and refinement analysis with GSAS-II, scanning electron microscope (SEM), Fourier transform infrared (FT-IR) spectra, UV–Vis reflectance spectrum (UV–Vis RS), and photoluminescence (PL) spectra, respectively. XRD revealed a pure tetragonal phase structure without any impurities of the as-prepared YVO4:Eu3+ microspheres. SEM images indicated that the self-assembled YVO4:Eu3+ architectures were made up of microspheres with the average diameter of around 300 nm, which were self-assembled from tiny packed nanocrystallites with 24–30 nm in diameter. Remarkably, the microstructure of these self-assembled microspheres could be well preserved during the calcination process at high temperatures. Under UV light irradiation, YVO4:Eu3+ microspheres exhibited a bright red emission corresponding to the 5D1→7F1 and 5D0→7FJ (J = 1, 2, 3, 4) transitions of the Eu3+ ions. More importantly, compared with the as-prepared YVO4:Eu3+ microspheres, the microspheres after the calcined treatment showed superior optical properties, as indicated by higher emission intensity, longer decay lifetime, and higher quantum efficiency. The present results showed that the self-assembly synthesis combined with the calcination treatment could provide a facile route to optimize the luminescent properties of inorganic functional materials.