In this Letter, we experimentally show that the single-photon fluorescence in IR144 solution can be enhanced by manipulating the laser spectral phase. We theoretically and experimentally validate that the single-photon fluorescence enhancement should be attributed to the non-resonant two-photon absorption from the state S-1 -> S-2. Finally, our proposed mechanism is further confirmed by repeating the SPF experiment in the low laser intensity, where the non-resonant two-photon absorption from the sate S-1 -> S-2 is eliminated. (C) 2010 Elsevier B. V. All rights reserved.