Photo-induced ring-opening reaction from flav-3-en-2-ol to 2-hydroxychalcone has been studied by time-resolved infrared (TR-IR) spectroscopy and quantum chemical calculations. A vibrational band due to the C═O stretching modes for intermediate species, enol forms of 2-hydroxychalcone in the electronic ground state, was observed at 1632 cm^–1 in the TR-IR spectra after photoexcitation of flav-3-en-2-ol. We also found that the C═O stretching modes of the keto forms of 2-hydroxychalcone at 1664 cm^–1 appeared immediately after photoexcitation and increased in intensity in synchronization with the depletion of the 1632 cm^–1 band. Because the decay of the 1632 cm^–1 band and the rise of the 1664 cm^–1 band were fitted with bi-exponential model functions with common rate constants 0.5 and 11 μs^–1, we propose that two kinds of enol form, single bond cis- (s-cis-) and trans- (s-trans-) enols, transformed into keto forms, cis-2-hydroxychalcone (Cc) and trans-2-hydroxychalcone (Ct), respectively. Quantum chemically calculated IR spectra of related species are consistent with the proposal. The observed temporal behavior of the TR-IR spectra indicates that there were reaction paths to the photogeneration of Cc and Ct within the time resolution of the TR-IR spectrometer (∼0.1 μs) in addition to the reaction paths via the enol forms of 2-hydroxychalcone.