An off–on fluorescence aptasensor was developed for trace thrombin detection based on fluorescence resonance energy transfer (FRET) between CdS QDs and gold nanoparticles (AuNPs). Using DNA pairwise hybridization of the aptamer to the complementary DNA (cDNA), the CdS QDs (energy donor) were tightly coupled to the AuNPs (energy acceptor), resulting in the occurrence of FRET and there was a dramatic fluorescence quenching of CdS QDs (turn off). When the thrombin was added to the fluorescence aptasensor, the specific binding of the aptamer to the target formed a G-quadruplex that caused the AuNPs receptor to detach and the DNA duplex to be disassembled. The process would inhibit the FRET which contribute to the recovery of fluorescence (turn on) and an “off–on” fluorescence aptasensor for thrombin detection was constructed accordingly. Under optimal conditions, the fluorescence recovery showed good linearity with the concentration of thrombin in the range of 1.35–54.0 nmol L⁻¹, and the detection limit was 0.38 nmol L⁻¹ (S/N = 3, n = 9). Importantly, the fluorescence aptasensor presented excellent specificity for thrombin, and was successfully applied to the quantitative determination of thrombin in real serum with satisfactory recoveries of 98.60–102.2%.