A series of water-soluble YVO₄:Bi³⁺,Eu³⁺ nanocrystals, with surfaces functionalized by a branch polyethylenimine (BPEI) polymer, have been synthesized via a one-pot hydrothermal method. It was found that the particle size and crystal morphology could be efficiently controlled by different reaction temperatures, pH values and molecular weights of the BPEI polymer. The surface modification of the nanocrystals was characterized using Fourier transform infrared spectroscopy (FT-IR). The highly crystalline YVO₄:Bi³⁺,Eu³⁺ nanoparticles, with an average diameter of 20 nm, can be dispersed in water due to the presence of amino ligands. When conjugated with biomolecules, the YVO₄:Bi³⁺,Eu³⁺ nanocrystals retain their strong red emission, peaking at 619 nm under near-ultraviolet (n-UV) excitation. The results indicate that YVO₄:Bi³⁺,Eu³⁺ nanocrystals can serve as a promising candidate for biological imaging, and immunoassay applications.