Fluorescence enhancement from a multilayered substrate fabricated with Ag and ${\mathrm{Al}}_2{\mathrm{O}}_3$ was investigated using fluorescein, rhodamine B, Cy3, and Cy5 as fluorophores. The change in the fluorescence enhancement with ${\mathrm{Al}}_2{\mathrm{O}}_3$ had two peaks and one valley in the range from 0 to $300\mathrm{nm}$ of ${\mathrm{Al}}_2{\mathrm{O}}_3$ thickness, and such peaks and valley were found to appear periodically. Moreover, the reflection of the excitation light from the multilayered substrate was investigated. The reflection of the excitation light periodically changed depending on the ${\mathrm{Al}}_2{\mathrm{O}}_3$ thickness as well, and the maximum reflection was observed near the ${\mathrm{Al}}_2{\mathrm{O}}_3$ thickness of the peak fluorescence enhancement. It was found that the periodic changes of the fluorescence enhancement and the reflection of the excitation light could be explained, for the most part, with the integral multiples of the $\lambda /4$ derived by a simple interference theory.