Durable ultraviolet (UV)-protective cotton fabric has great application potential in outdoor cotton clothing. In this study, oppositely charged TiO2 nanoparticles were deposited onto cotton fabric through the layer-by-layer self-assembly technique, resulting in multilayer films with UV-protective properties. The mechanism of the technology has been investigated through characterization of the structure and properties using different techniques including FTIR, UV–Vis spectroscopy, and a scanning electron microscope with an energy-dispersive X-ray spectrum. The results showed that TiO2 nanoparticles distributed uniformly on the surface of cotton fibers. The TGA results indicated that the TiO2 nanoparticles deposit on cotton fabrics had little effect on the thermal stability of cotton fabrics. The tensile strength and air permeability of the cotton samples were tested by a universal material testing machine and automatic ventilation instrument. The UV protection property of cotton fabric after assembled multilayer films was measured by an ultraviolet transmittance analyzer, and the laundering experiments were carried out to determine the durability of TiO2 nanoparticles on cotton fabric. The results showed that the UV protection property of cotton fabrics after assembled TiO2 nanoparticles was still maintained at a high level after five launderings.