Advancements in the fabrication of upconversion nanoparticles (UCNPs) for synthetic control can enable a broad range of applications in biomedical systems. Herein, we experimentally verified the role of the hydrothermal reaction (HR) time in the synthesis of NaYF:20%Yb/3%Er UCNPs on their morphological evolution and phase transformation at different temperatures. Characterizations of the as-prepared UCNPs were conducted using X-ray diffraction (XRD), electron microscopy and spectroscopy, and thermogravimetric and upconversion (UC) luminescence analysis. We demonstrated that determining the optimal HR time, also referred to here as the threshold time, can produce particles with good homogeneity, hexagonal phase, and UC luminescence efficiency. Subsequently, the polymer coated UCNPs maintained their original particle size distribution and luminescence properties, and showed improved dispersibility in a variety of solvents, cellular nontoxicity, in vitro bioimaging, and biocompatibility as compared to the bare UCNP. Besides this, polyacrylic acid conjugated UCNPs (UCNP@PAA) also revealed the strong anticancer effect by conjugating with doxorubicin (DOX) as compared to the free DOX. Based on these findings, we suggest that these particles will be useful in drug-delivery systems and as in vivo bioimaging agents synchronously.