The effects of Yb, Er on structural, optical, magnetic and biocompatibility of β-NaYF 4 compounds were examined. The pure and Yb, Er-doped β-NaYF 4 compounds were synthesized via simple hydrothermal method. X-ray diffraction analysis revealed the hexagonal crystal structure. The functional group present in the sample was identified by FT-IR analysis. The vibrational bands were analysed by Raman spectroscopy. Electron Microscope images exhibit the hexagonal plate-like morphology. The characteristic absorbance peaks of Yb, Er and host were obtained in UV-VIS-NIR absorbance spectra. The interesting green and enhanced red emission through Photoluminescence spectra of the prepared compounds might be useful for bio-imaging. Pure β-NaYF 4 possessed diamagnetic property and Yb, Er-doped β-NaYF 4 exhibited paramagnetic property at room temperature. Yb, Er-doped β-NaYF 4 was incubated with HeLa cells and assessed cytotoxicity using MTT assay. Hence, Yb, Er-doped β-NaYF 4 materials have promising application in cell imaging and bio-separation applications.