This paper identified a new excited-state proton transfer (ESPT) mechanism for 2,6-diazaindoles (2,6-DAI) in aqueous (H2O) solution based on time dependent density functional theory. The calculated results show that the excited-state three proton transfer reaction cannot occur because the 2,6-DAI with two water molecules do not form hydrogen bond wires; this finding was different from those reported in previous experiments (Chung et al. J. Am. Chem. Soc. 2017, 139, 6396-6402). 2,6-DAI with three water molecules form 2,6-DAI.(H2O)(3) dusters, whereas 2,6-DAI with four water molecules form 2,6-DAI.(H2O)(4) cluster. These dusters participate in the ESPT reaction. To determine the ESPT mechanism of 2,6-DAI.(H2O)(3) and 2,6-DAI.(H2O)(4) dusters, we constructed the potential energy curves of S, and So states. The results confirmed the simultaneous presence of both 2,6-DAI.(H2O)(3) and 2,6-DAI. (H2O)(4) dusters and only one proton transfer pathway. By calculating the transition states of 2,6-DAI.(H2O)(3) and 2,6-DAI.(H2O)(4) clusters, we found that the ESPT reaction is a consistent mechanism. Our work investigated the number of water molecules involved in the ESPT and paved the way to further study the intermolecular hydrogen bonding interactions in the biological field.