Monodisperse NaYF₄ : Yb, Er upconversion fluorescent nanoparticles were firstly synthesized via a co-precipitation method in the presence of diethylenetriamine pentoacetic acid(DTPA). The nanoparticles were characterized by using of X-ray diffraction (XRD), transmission electron microscope (TEM), fluorescence(FL) spectrum, and thermogravimetry-differential scanning calorimetry (TG-DSC) analysis. The as-prepared nanoparticles were uniform, and their size could be controlled in a range of 20 to 120 nm by varying the amount of DTPA. During the precipitation reaction, DTPA molecules could form a complex with the rare earth ions, and then the rare earth ions were released slowly to react with F^－ions, which slowed down the speed of the reaction. In addition, DTPA molecules could also be capped on surface of the growing nanoparticles, which prevented the nanoparticles from aggregation. After annealing, the nanoparticles were transformed from cubic phase to hexagonal phase, and their upconversion fluorescence intensity was enhanced remarkably. The synthesis conditions including the amount of chelating agents and temperature for annealing, which showed great influence on the size, phase and upconversion fluorescence intensity of the nanoparticles, were also discussed. It was confirmed by XRD and TG-DSC analysis that the presence of DTPA suppressed the cubic-to-hexagonal phase transition of the nanoparticles. However, while the small nanoparticles were obtained with well control, the annealed crystals synthesized by adding DTPA could still emit strong fluorescence under a low exciting power, which could well fulfill the demand for biolabeling. These nanoparticles are envisioned to find potential applications in biological detections.