Symmetric multipolar (e.g., quadrupolar and octupolar) chromophores that undergo excited-state symmetry breaking in polar solvents have been drawing considerable attention for a long time as promising two-photon absorption and luminescent materials. However, this process has been generally restricted to liquid media. Herein, we report the excited-state symmetry breaking of a centrosymmetric D−π–D (donor−π–donor) quadrupolar molecule, DB2, occurring at a polymer/glass interface. In polar liquids, DB2 exhibits pronounced emission solvatochromism originating from symmetry-breaking intramolecular charge transfer in the S1 state. Single-molecule fluorescence spectroscopy shows that the fluorescence characteristics of DB2 at the interface are strikingly different from those of DB2 embedded in a polymer film, that is, the dramatic lengthening of fluorescence lifetimes and pronounced sharpening of fluorescence spectra are observed. With the aid of theoretical calculations, it is revealed that these characteristic behaviors are due to the strong electronic localization of the emitting excited state via asymmetric in-plane relaxation of the π-conjugated framework through dipolar interactions with the glass surface.