We report total internal reflection fluorescence microscopy (TIRFM) measurements using an atomic force microscope (AFM) probe to investigate the quantitative fluorescence profiles of CdSe/ZnS nanocrystals (NCs), which interact with gold surfaces, as a function of the gold–NC distance. First, a silica bead was glued to an AFM cantilever. CdSe/ZnS NCs were then immobilized on the silica-bead-functionalized AFM probe for control of the gold–NC distance, while a gold thin film served as the metal surface on a glass substrate. As a result of coexistent fluorescence resonance energy transfer and localized surface plasmon resonance (LSPR) coupling, a strong enhancement was observed at 15 nm, while fluorescence suppression was seen at a proximal distance. For further quantitative discussions of the effects of the metal surface on the fluorescence behavior of single NCs, we introduced a gold-deposited silica bead onto the AFM cantilever to regulate the gold–NC distance. Subsequently, the CdSe/ZnS NCs were immobilized on a glass surface. As a result of compensation for the contribution of photobrightening, the NC fluorescence profile for the glass surface also clearly displayed the LSPR-coupled enhancement at 10 nm. In contrast, it was discovered that positioning of the gold surface at a proximal single NC gave rise to quenching of the NC fluorescence. Our findings suggest that a method using an NC-functionalized AFM probe allows us to quantitatively investigate the NC fluorescence under the effects not only of a metal surface but also of an evanescence field.