Exosomes are often a promising source of biomarkers for cancer diagnosis in the early stages. Therefore, it is important to develop a sensitive and low-cost detection method. Here, we introduce a new substrate using gold nanorods (GNRs) on a silver-island film that produces a 360-fold AF647 molecule fluorescence enhancement compared to glass. The amplified fluorescence was proven theoretically by using finite difference time-domain simulation (FDTD). Utilizing the enhanced fluorescence from the substrate, GNRs attached with the biomolecules and created a sandwich immunoassay that can significantly detect human CD63 antigen on the exosome. By applying the method, the detection limit of mouse IgG goes down to 0.3 ng/mL, which is considerably better than the existing methods. Moreover, the sensitivity and accuracy for clinical plasma from six patients confirm its diagnostic feasibility. The proposed substrate can be uniformly extended to the identification of other biomarkers by modifying the antibodies on the surfaces of the GNRs.