The synergistic effects of deep cryogenic treatment (DCT) and the ultrasonic surface rolling process (USRP) on surface microstructure and properties of Ti6Al4V alloy were investigated. Surface roughness, surface topography, nano-hardness and residual stress were used to characterize the surface-modified layer. A plastic deformation layer with a deformation depth of 7.80 μm was formed in the DCT + USRP specimen. The content of the β phase in the surface of the DCT + USRP specimen was 3% less than that of USRP, making the surface hardness of the DCT + USRP specimen higher than that of USRP. Further, the surface roughness of the DCT + USRP specimen was lower. Especially, tension-tension fatigue tests showed that the fatigue life of the specimen after DCT + USRP treatment increased by 61.9% relative to the original specimen. The synergy mechanisms of surface quality, residual stress, microstructure and work hardening were analyzed in the DCT + USRP specimen.