The denaturation of double-stranded deoxyribonucleic acid (ds-DNA) has been well known to break nucleobase bonds, resulting in single-stranded deoxyribonucleic acid (ss-DNA) in solutions, which can recombine to form ds-DNA in a reversible manner. We developed an efficient process to irreversibly maintain various DNA denaturation levels in thin solid films in order to investigate the impacts of the denaturation on the optical properties of DNA films. By adding NaOH in an aqueous solution of salmon testis DNA, we flexibly controlled the level of denaturation in the solution, which was then spin-coated on Si and silica substrates to irreversibly bind ss-DNAs in a thin solid film. The denaturation of DNA in thin solid films was experimentally confirmed by ultraviolet-visible and Fourier transform infrared spectroscopic investigations, whose level could be controlled by the NaOH content in the aqueous solution precursor. By this irreversible denaturation process, we developed a new method to flexibly vary the refractive index of DNA thin solid films in a wide range of Δn