As artificial receptors for protein recognition, epitope-imprinted polymers combined with fluorescence sensing based on quantum dots (QDs) can be potentially used for biological analysis and disease diagnosis. However, the usual way for fabrication of QD sensors through unoriented epitope imprinting is confronted with the problems of disordered imprinting sites and low template utilization. In this context, a facile and efficient oriented epitope surface imprinting was put forward based on immobilization of the epitope templates via thiol-disulfide exchange reactions. With N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as a heterobifunctional reagent, cysteine-modified epitopes of cytochrome c were anchored on the surface of pyridyl disulfide functionalized silica nanoparticles sandwiching CdTe QDs. After surface imprinting via a sol-gel process, the epitope templates were removed from the surface-imprinted layers simply by reduction of the thiol-disulfide, affording oriented epitope-imprinted sites. By this method, the amount of epitope templates was only 1/20 of traditionally unoriented epitopes. The resulting sensors demonstrated significantly enhanced imprinting performance and high sensitivity, with the imprinting factor increasing from 2.6 to 3.9, and the limit of detection being 91 nM. Such epitope-oriented surface-imprinted method may offer a new design strategy for the construction of high-affinity protein recognition nanomaterials with fluorescence sensing.