A novel sensitive aptasensor for vascular endothelial growth factor-165 (VEGF₁₆₅) was constructed based on fluorescence resonance energy transfer (FRET) by employing upconversion nanoparticles (UCNPs) and MoS₂ nanosheets as the energy donor and acceptor, respectively. The upconversion fluorescence resonance energy transfer (UC-FRET) was triggered by the physical adsorption interaction between the aptamer and MoS₂ nanosheets, leading to a remarkable quenching of UCNP fluorescence up to 95%. Upon addition of VEGF₁₆₅ to the UCNP–aptamer system before MoS₂ nanosheets were added, the aptamer preferentially bound to VEGF₁₆₅ with the change of spatial conformation, which weakened the van der Waals' force between the MoS₂ nanosheets and the aptamer, thus leading to the separation of the donor and the acceptor. Consequently, the FRET phenomenon was inhibited and the luminescence of UCNPs was regained, which was linearly related to the concentration of VEGF₁₆₅ in the range of 0.1 ng mL⁻¹ to 16 ng mL⁻¹. By taking advantage of the extreme fluorescence quenching ability of MoS₂ nanosheets and the optical merits of UCNPs, the aptasensor based on UC-FRET exhibited favorable performance for the homogeneous assay of VEGF₁₆₅ in human serum, which is of great value for clinical diagnosis of tumors and related biological studies.