Lanthanide nanoparticles (NPs), characterized by their large Stokes’ shifts, narrow emission bands and high photochemical stability, are promising candidates for optical probes in biological research. As a typical red emission material, Eu3+-doped YVO4 is investigated in pursuit of an integral methodology for Ln-based bioprobes. Assisted with phosphino-polyacrylic acid, YVO4:Eu NPs with a luminescent quantum yield of ca. 54% are synthesized via a one-pot hydrothermal reaction. The obtained NPs exhibit good water/buffer stability and feasible biomodification benefiting from the surface carboxylic acid groups. However, having an inorganic core of ca. 20 nm, these NPs do not affect the conformation of surface conjugated proteins (bovine serum albumin), as confirmed with the circular dichroism spectrum. A specific recognition protocol is demonstrated by biotinated YVO4:Eu NPs in quantitative fluoroimmunoassay and microchip assay. Furthermore, the biocompatibility of the Ln NPs, which is an important aspect towards biological application, is demonstrated with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Cell imaging studies conducted with spectral mapping technique and confocal fluorescent microscopy demonstrate the YVO4:Eu NPs to be a type of competitive luminescent probes for in vitroapplications.