Abstract: Effects of hydrogen bonds on two-photon absorption (TPA) of a new donor-acceptor type green fluorescent protein chromophore analogue are investigated by employing a combined molecular dynamics and quantum chemistry method. The probable configurations of the chromophore in water are extracted from molecular dynamics simulation and the TPA properties of more than twenty hydrogen bond complexes are computed by quadratic response theory. Thereby, the structure and property relations are established. Three types of hydrogen bonds including O···H−O, N−H···O and N···H−O can be formed between the chromophore and water molecules. The O···H−O induces a little decrease of TPA cross section with a red-shifted wavelength. The N−H···O gives rise to a great enhancement of TPA at a longer wavelength, while the N···H−O decreases TPA significantly and makes the wavelength blue-shifted. The reasons for these effects are rationalized well by using a two-state model analysis. The related molecular orbitals are also plotted to visualize the charge transfer characters. In addition, the averaged TPA spectrum is obtained by calculating the probabilities of various hydrogen bond complexes. Our research could provide a good insight into the design of two-photon materials by making use of hydrogen bond networks.