Elucidating the mechanism and estimating the extent of conformation change of double-stranded DNA (dsDNA) upon ultraviolet (UV) exposure are of vital importance for understanding the DNA photodamage process. The existing research was mainly focused on the lesions of single-stranded DNA (ssDNA) and involved off-site measurement of the photodamage level. In this work, short-wavelength UV (UVC) (254 nm) irradiation was demonstrated to induce the dehybridization of dsDNA due to the loss of paring capacity of photodamaged pyrimidine nucleobases. The intrinsic programmability of dsDNA enabled researchers to rationally design the on-demand dehybridization sites. The spatial conformation switch of dsDNA caused by UVC irradiation could be evolved into a label-free sensing platform for the on-site measurement of the DNA photodamage level.