Dense multiscale silica (SiO2) micro- and nanostructures are fabricated on a pre-stressed polymer film. This novel SiO2 substrate serves as a robust platform to enhance the fluorescence signal of bound biomolecules. Through a combination of surface concentration, light scattering, and changes in the photophysical properties of the confined dye molecules, dramatic fluorescence signal enhancements (average = 116 times greater than on planar glass) and increased signal-to-noise ratio (76:1) are demonstrated with tetramethylrhodamine isothiocyanate (TRITC)-conjugated streptavidin (STRITC) on SiO2 structures. Enhanced detection sensitivity of STRITC over glass on the SiO2 structures is achieved down to a detection limit of 11 ng mL−1. Such significant fluorescence signal enhancements have importance in practical applications such disease diagnostics and surface sensing.