In this paper, we introduce the concept of a “polymeric ruler” for investigating distance-dependent emission behaviors of fluorophores, namely the quenching or enhancement of fluorescence, on flat Au surfaces in the range of 5 to ∼100 nm, which has not previously been easily accessible. The polymeric ruler is constructed by a highly controllable surface-initiated atom transfer radical polymerization of oligo(ethylene glycol)methacrylate (OEGMA), and the obtained thicknesses of the poly(OEGMA) (pOEGMA) layers range from ∼5 to ∼80 nm. The quenching or enhancement of fluorescence is found to be dependent upon the distance between fluorophores and the Au surface. In brief, fluorescence quenching occurs at distances within about 15 nm from the Au surface, and surface-enhanced fluorescence is observed at tens of nanometers beyond the range of quenching with the maximum enhancement at about 40–50 nm. The obtained information on the distance-dependent surface-enhanced fluorescence is applied to the construction of highly sensitive bioassay platforms: the use of the 50 nm thick pOEGMA layer lowers the detection limit up to 1 pM.