We present a method to modulate the fluorescence of non-polar single squaraine-derived rotaxanes molecules embedded in a polar poly(methyl methacrylate) (PMMA) matrix under an external electric field. The electron transfer between single molecules and the electron acceptors in a PMMA matrix contributes to the diverse responses of fluorescence intensities to the electric field. The observed instantaneous and non-instantaneous electric field dependence of single-molecule fluorescence reflects the redistribution of electron acceptors in PMMA induced by electronic polarization and orientation polarization of polar polymer chains in an electric field.