In recent years, the excellent performance of deep eutectic solvents (DES) has garnered increasing attention in the pursuit of sustainable solvents. This study employs natural and biodegradable kojic acid (KA) as a hydrogen bond donor (HBD) and environmentally friendly urea derivatives as hydrogen bond acceptor (HBA) to construct a series of novel type V KA/urea derivatives DESs. The as-obtained DESs were systematically characterized by density, viscosity, refractive index, speed of sound, thermal gravimetric analysis, FT-IR and NMR spectrums. The behavior of components in the DESs were deduced on the basis of thermal expansion coefficient (αP), apparent molar volume (V2∅), free volume (Fm), and spectroscopic analysis. The interactions in the DESs were further cross-checked by Quantum chemical calculation and molecular dynamics simulation. The consistent sequence of interaction strength in the DESs was TMU-KA > DMI-KA > DMPU-KA. For KA molecule, the hydroxyl H on ring displayed lower activity than that on the side-chain due to the dual impact of conjugation and volume repulsion effect. The obtained information can provide help for understanding structure-property relationship and exploring potential utilization for present KA/Urea DESs.