Lanthanide-doped upconversion nanoparticles with a suitable surface coating are appealing for biomedical applications. Because high-quality upconversion nanoparticles are typically prepared in an organic solvent and passivated by hydrophobic oleate ligands, a convenient and reliable method for the surface modification of upconversion nanoparticles is thus highly desired to satisfy downstream biological investigations. In this work, we describe a facile and versatile strategy for displacing native oleate ligands on upconversion nanoparticles with a diversity of hydrophilic molecules. The ligand-exchange procedure involves the removal of original oleate ligands followed by the attachment of new ligands in a separate step. The successful coating of relevant ligands was confirmed by Fourier transform infrared spectroscopy, thermogravimetry analysis, and ζ-potential measurement. The surface-modified nanoparticles display high stability and good biocompatibility, as revealed by electron microscopy, photoluminescence spectroscopy, and cytotoxicity assessment. Our study demonstrates that functional biomolecules such as biotin can be directly immobilized on the nanoparticle surface using this approach for the quick and effective detection of streptavidin.