DNA’s structure stability in hydrated deep eutectic solvents (DESs) is getting growing attention for emerging bio-applications. The employment of DESs as novel co-solvents in water media could favor eco-friendly and biodegradable materials for DNA storage and handling. Understanding the molecular interactions between nucleic acids and aqueous DES is crucial for developing new-generation solvents for biomolecules. In this work, we exploit the molecular sensitivity and selectivity of synchrotron radiation UV resonance raman (SR-UVRR) spectroscopy to explore the interplay between a choline chloride:urea (ChCl:U) DES and double-stranded DNA. Our study analyzes the impact of ChCl:U on the DNA’s thermal unfolding pathway by focusing on the guanine nucleobases whose Raman signal could be strongly enhanced through careful tuning of the excitation wavelength.