To achieve sensitive biomolecular detection, a gold nanohole array sensing chip was designed by plasmonic simulation and fabricated by high fidelity nanoimprinting technology. The gold nanohole array on a glass substrate serves as an optical antenna array, to produce localized surface plasmon resonance (LSPR) of 10–100 times stronger than the illumination light for quantum dot (QD) excitation. In this work, a robust sandwich bioassay of primary aptamer/thrombin/secondary aptamer with QD655 was established on the chip, to detect thrombin at a limit of detection (LOD) of 1ng/ml (27pM), with low non-specific binding even at the presence of 1000 times more concentrated bovine serum albumin (BSA). The conformation and QD enhancement mechanism of the sandwich assay were characterized by atom force microscopy (AFM) and dual polarization interferometry (DPI). This is the first time that DNA is surface functionalized on gold nanostructures for protein detection by plasmon enhanced QD emission. Considering the low-cost, easy regeneration and long shelf-life of the capturing aptamer, low non-specific binding and high sensitivity of the bioassay, as well as the inexpensive mass fabrication of the high quality chips, this novel aptasensor platform is particularly useful for a point-of-care system on medical diagnostics.