Given the continuously increasing requirements for biological detection, upconversion nanoparticle (UCNP)-based techniques offer a novel research direction for biomolecule detection. Due to their superior and stable photophysical characteristics, UCNPs have received widespread attention and have been extensively applied for reliable and highly sensitive detection of biomolecules. Herein, we review articles based on three aspects: UCNP synthesis, surface functionalization, and application in biomolecule detection. The future development trend of UCNPs is also anticipated. Several synthesis methods are introduced, and the advantages and disadvantages of these synthesis methods are summarized and compared. Control of UCNP size and shape, as well as UCNP doping, is discussed. For surface modification, we introduce approaches such as surface silanization, ligand exchange, ligand oxidation, ligand attraction and electrostatic layer by layer assembly. In addition, we focus on UCNP-based luminescence sensors for the analysis of biomolecules, such as nucleic acids, amino acids, proteins, carbohydrates, uric acid and lipids. These upconversion nanomaterials were used as carriers for recognition, transmission or signal amplification and significantly improved the performance of biomolecule detection. Finally, the shortcomings of UCNP are analyzed and its future prospects are presented. This review provides new insights into the application of upconversion nanoparticles as an important element in biomolecule detection.