The systematic evolution of ligands by exponential enrichment (SELEX) is a selection process for identifying high-affinity selective molecules from a randomized combinatorial nucleic acid library against a wide range of target molecules. Using a pool of N25 RNA molecules, the SELEX process was performed against two targets from influenza viruses, namely, intact influenza B/Tokio/53/99 and hemagglutinin of infuluenza B Jilin/20/2003. The selection processes were evaluated by surface plasmon fluorescence spectroscopy (SPFS), and the result was compared to that obtained by a conventional radioisotope method. Clear discrimination among different selection cycles was displayed by SPFS, indicating that this method can be used as an alternative method of radioisotope labeling. The dissociation constant of the selected aptamers against the targets was in the low nanomolar range. The sensitivity of the selected aptamer against intact influenza B/Tokio/53/99 to detect the influenza virus was the low ng/mL level, an approximately 250-fold higher sensitivity than that of the commercially obtained antibody. The target binding sites on the aptamer were predicted by mapping analyses. The selected aptamer could discriminate other influenza strains, and the sensitivity of the selected aptamer was further confirmed by gold-nanoparticle-based sensing on a waveguide-mode sensor. This finding demonstrates that the selected aptamer would be useful for detecting influenza viruses at an early stage of infection and for the purpose of influenza surveillance.