A new luminescence energy transfer (LET) system has been designed for the detection of thrombin in the near-infrared (NIR) region by utilizing NIR-to-NIR upconversion lanthanide nanophosphors (UCNPs) as the donor and gold nanorods (Au NRs) as the acceptor. The use of upconverting NaYF4:Yb3+,Tm3+ nanoparticles with sharp NIR emission peaks upon NIR excitation by an inexpensive infrared continuous wave laser diode provided large spectral overlap between the donor and the acceptor. Both the Au NRs and carboxyl-terminated NaYF4:Yb3+,Tm3+ UCNPs were first modified with different thrombin aptamers. When thrombin was added, a LET system was then formed because of the specific recognition between the thrombin aptamers and thrombin. The LET system was used to monitor thrombin concentrations in aqueous buffer and human blood samples. The limits of detection for thrombin are as low as 0.118 nM in buffer solution and 0.129 nM in human serum. The method was also successfully applied to thrombin detection in blood samples.