A sandwich enzyme-linked dendrimer-aptamer assay (ELDAA) has been developed using glass-bottom 96-microwell plates in order to improve the detection performances of a traditional enzyme-linked immunosorbent assay (ELISA). To this end, poly(amidoamine) (PAMAM) dendrimers were chemically immobilized onto glass-bottom surfaces of the microwells, and aptamers against a model analyte, i.e., human platelet-derived growth factor BB (PDGF-BB), were subsequently conjugated onto the microwell surfaces via the immobilized PAMAM dendrimer templates. These layer-by-layer surface modifications were characterized using fluorescence microscopy, water contact angle, X-ray photoelectron spectroscopy, and atomic force microscopy to confirm the success of each surface modification steps. Moreover, the effects of different PAMAM dendrimer templates on the overall performances of the ELDAA were also studied. Our results showed that the new ELDAA had a much broader linear detection range that was approximately 2 times of that of a commercial ELISA and a significantly lowered limit of detection that was about 4.5 more sensitive in comparison with the standard ELISA. It can be concluded that the new ELDAA offers more versatile detection performances than the traditional ELISA technique and can be readily adapted into existing ELISA workflow, therefore offering a promising and robust alternative to the ELISA technique.