A homogeneous immunoassay for the sensitive and selective determination of trace amounts of a-fetoprotein (AFP, a cancer marker) by detection in the near-infrared (NIR) region based on luminescence energy transfer (LET) from NaYF4:Yb,Tm/NaGdF4 core-shell upconverting nanoparticles to gold nanorods (GNRs) is presented. The carboxyl-functionalized NaYF4:Yb,Tm/NaGdF4 core-shell upconverting nanoparticles (UCNPs) were excited by a 980 nm continuous wavelength laser, and its emission peak appeared at a near-infrared wavelength (similar to 804 nm). The carboxyl-functionalized upconverting nanoparticles were conjugated with the anti-AFP (Ab1) and acted as donor. GNRs with a high absorption band around 790 nm, which was overlapped the UCNPs emission, were synthesized and acted as the acceptor. The donor (negatively charged) interacted with the acceptor (positively charged) via electrostatic interactions to bring them into close proximity. LET could occur, producing a quenching phenomenon. When the AFP antigens were added into the system, the binding affinity between AFP and Ab1 was stronger than the electrostatic interactions, which released the energy acceptors from the energy donors, interrupting luminescence energy transfer, and therefore, the luminescence was recovered. On the basis of the restored luminescence, a turn-on optical immunosening system was developed. Under the optimal conditions, the linear range of detection was from 0.18 to 11.44 ng/mL for AFP (R = 0.99), with a detection limit as low as 0.16 ng/mL. The proposed method has also been used to monitor AFP in human serum samples. Therefore, further study based on the NaYF4:Yb,Tm/NaGdF4 core-shell nanoparticles-GNRs construction may open the way for a new class of NIR-LET biosensors with wide applications.