In this study, the optimal condition of a silver (Ag) film deposited on a cover slip for surface plasmon-coupled emission (SPCE) induced two-photon excited fluorescence (TPEF) based on an objective-based, total internal reflection (TIR) microscope was investigated. According to the theoretical simulations of local electric field enhancement and fluorescence coupled emission efficiency, the thickness of the Ag film should be about 40 nm in order to maximize the TPEF collection efficiency by the objective. The deposited Ag film with a germanium seed layer on a cover slip exhibits additional improvement in surface smoothness by reducing variations in surface roughness to below 1.0 nm, thereby reduces local hot spots which degrade the image uniformity. Moreover, an Ag film with a 20 nm-thick SiO2 spacer not only prevents damage caused through interaction with the aqueous solution under high laser power irradiance, but also reduces the fluorescence quenching effect by the Ag film. By optimizing the Ag film thickness, surface smoothness, and a protective dielectric spacer, efficient TIR TPEF imaging can be achieved through SPCE.