Femtosecond ultrafast electronic relaxation and vibrational dynamics in 2'-hydroxychalcone after deep ultraviolet (DUV) excitation were observed by two types of pump-probe spectroscopy experiments, i.e., DUV-pump pulse and visible-broadband-probe pulse (DUV/Vis) experiments and DUV-pump and DUV-probe (DUV/DUV) pulse experiments. Time-dependent density functional theory (TDDFT) calculations were performed to elucidate relaxation dynamics from the third singlet electronic excited state S3. The DUV/Vis experiments and TDDFT calculations have disclosed the ultrafast dynamics of internal conversion from the initial S3 state (τ1 ≈ 35 fs) to the S1 state via a rapid process through the S3/S2 conical intersection and proton transfer [OH: τ2(H) ≈ 93 and OD: τ2(D) ≈ 164 fs] before deactivation through the S1/S0 conical intersection (τ3 ≈ 690 fs). Thanks to the ultrashort pump and probe pulses, real-time observation of vibrational modes coupled to the electronic excitation was realized providing both amplitudes and phases. Spectrogram analyses were performed based on the real-time spectra obtained by the DUV/Vis experiments, in which instantaneous vibrational frequencies reflecting molecular structural change after the impulsive excitation were visualized. The vibrational frequency of central C[double bond, length as m-dash]C bond stretch decreases from ∼1600 cm-1 to ∼1560 cm-1 in about 200-500 fs and recovers in ∼550 fs. Normal mode analyses along the decay path support the observed variation of the C[double bond, length as m-dash]C stretching frequency. The temporal weakening of the central C[double bond, length as m-dash]C bond is connected with the angle of the two aromatic rings which flip back to the initial conformation.