We report an immunosensor based on luminescence resonance energy transfer (LRET) to detect homogeneous glycated hemoglobin (HbA1c). This system uses near-infrared (NIR)-to-visible rare-earth upconversion nanoparticles (UCNPs), such as NaYF4:Yb(3+), Er(3+), as the donor and HbA1c as the acceptor. The HbA1c used as target molecules showed absorption at 541 nm, which corresponded with the emission of the UCNPs. When HbA1c was added, LRET occurred between the donor and acceptor under laser irradiation of 980 nm because of the specific recognition between the anti-HbA1c monocolonal antibody-functionalized UCNPs and HbA1c. In the absence of HbA1c, there was strong upconversion luminescence intensity; however, in its presence, the distance between the donor and acceptor decreased to enable energy transfer, consequently quenching the luminescence of the UCNPs. The proposed method was successfully applied to HbA1c detection in blood samples. Our results indicate that the LRET-based immunosensor allows for specific and sensitive detection of HbA1c in a homogeneous manner.