We report high-resolution spectroscopic detection of TiO molecular signature in the day-side spectra of WASP-33b, the second hottest known hot Jupiter. We used the High Dispersion Spectrograph (HDS; R ∼ 165,000) on the Subaru telescope in the wavelength range of 0.62-0.88 m to obtain the day-side spectra of WASP-33b. We suppress and correct the systematic effects of the instrument and the telluric and stellar lines using the SYSREM algorithm after the selection of good orders based on Barnard's star and other M-type stars. We detect a 4.8 signal at an orbital velocity of km s−1 and systemic velocity of km s−1, which agree with the derived values from a previous analysis of the primary transit. Our detection with the temperature inversion model implies the existence of a stratosphere in its atmosphere; however, we were unable to constrain the volume mixing ratio of the detected TiO. We also measure the stellar radial velocity and use it to obtain a more stringent constraint on the orbital velocity, km s−1. Our results demonstrate that high-dispersion spectroscopy is a powerful tool to characterize the atmosphere of an exoplanet, even in the optical wavelength range, and shows a promising potential in using and developing similar techniques with high-dispersion spectrograph on current 10 m class and future extremely large telescopes.