•MPEG-b-PMAA-YVO4:Eu provided lower cytotoxicity than many inorganic imaging agents.•A defined model was established by analysising the coefficients of pH and CEu%.•The synthesis under the direction of model achieved optimal size and fluorescence.•The strategy composed by block copolymer capping RENPs was benefit to bio-imaging.A novel nanoparticle with multilayer core–shell architecture for cell imaging is designed and synthesized by coating a fluorescent YVO4:Eu core with a diblock copolymer, MPEG-b-PMAA. The synthesis of YVO4:Eu core, which further makes MPEG-b-PMAA-YVO4:Eu NPs adapt for cell imaging, is guided by the model determined upon the evaluation of pH and CEu%. The PMAA block attached tightly on the YVO4:Eu core forms the inner shell and the MPEG block forms the biocompatible outermost shell. Factors including reaction time, reaction temperature, CEu% and pH are optimized for the preparation of the YVO4:Eu NPs. A precise defined model is established according to analyzing the coefficients of pH and CEu% during the synthesis. The MPEG-b-PMAA-YVO4:Eu NPs, with an average diameter of 24 nm, have a tetragonal structure and demonstrate luminescence in the red region, which lies in a biological window (optical imaging). Significant enhancement in luminescence intensity by MPEG-b-PMAA-YVO4:Eu NPs formation is observed. The capping copolymer MPEG-b-PMAA improves the dispersibility of hydrophobic YVO4:Eu NPs in water, making the NPs stable under different conditions. In addition, the biocompatibility MPEG layer reduces the cytotoxicity of the nanoparticles effectively. 95% cell viability can be achieved at the NPs concentration of 800 mg L−1 after 24 h of culture. Cellular uptake of the MPEG-b-PMAA-YVO4:Eu NPs is evaluated by cell imaging assay, indicating that the NPs can be taken up rapidly and largely by cancerous or non-cancerous cells through an endocytosis mechanism.