To improve the yield strength of titanium alloy Ti-6Al-4V material by ultrasonic burnishing, ultrasonic amplitude was used as the unique variable in this work. Four groups of experiments were set to analyze the stress-strain behavior of titanium alloy Ti-6Al-4V material on the machined surface and 30-50 μm below the surface. Ultrasonic amplitude in four groups was set as 0, 5, 7 and 10 μm, respectively. The burnishing of Ti-6Al-4V material without ultrasonic vibration was adopted as the control group. Two-phase distributions of titanium alloy Ti-6Al-4V material on the machined surface and 30-50 μm below the surface were measured by X-ray diffractometer (XRD). Scanning Electron Microscope (SEM) was used to analyze the plastic deformation degree of titanium alloy Ti-6Al-4V grains in the machined surface layer at different ultrasonic amplitudes. Energy Dispersive Spectrometer (EDS) was applied to observe the composition element distribution of the burnished titanium alloy Ti-6Al-4V surface layer under different ultrasonic amplitudes. Universal testing machine was used to obtain the various stress-strain curves of the burnished titanium alloy Ti-6Al-4V samples at different ultrasonic amplitudes. Lastly, the change rules of stress-strain behavior, plastic deformation and two-phase distribution for the burnished titanium alloy Ti-6Al-4V under different ultrasonic amplitudes were comparatively analyzed. The effect of microstructure on the stress-strain behavior for the ultrasonic burnished titanium alloy Ti-6Al-4V was clarified. Results indicated that the plastic deformation degree of titanium alloy Ti-6Al-4V grain increased with ultrasonic amplitude. The volume fraction of β phase for the ultrasonic burnished titanium alloy Ti-6Al-4V decreased firstly and then increased with the increase of ultrasonic amplitude. When ultrasonic amplitude was 7 μm, the volume fraction of β phase on the burnished titanium alloy Ti-6Al-4V surface reached the maximum value 19.70%. The volume fraction of β phase in the titanium alloy Ti-6Al-4V surface layer decreased along the depth during ultrasonic burnishing. However, stable element Al for α phase and stable element V for β phase did not show the obvious trend of the same rules with the increase of ultrasonic amplitude. After ultrasonic burnishing, the yield strength of titanium alloy Ti-6Al-4V showed a trend of decrease firstly and then increased with the increase of ultrasonic amplitude. When ultrasonic amplitude increased from 5 μm to 10 μm, the yield strength value of the ultrasonic burnished Ti-6Al-4V material was 1.06 GPa, 1.03 GPa and 1.16 GPa, respectively. Compared to the yield strength value 0.91 GPa of titanium alloy Ti-6Al-4V burnished without ultrasound, the yield strength of the ultrasonic burnished Ti-6Al-4V increased by 16.48%、13.19% and 27.47%, respectively. When ultrasonic amplitude was 10 μm, the yield strength of titanium alloy Ti-6Al-4V material reached the maximum. The increase of ultrasonic amplitude can increase the plastic deformation degree of the burnished titanium alloy Ti-6Al-4V material. Appropriate ultrasonic amplitude can change the two-phase distribution of the ultrasonic burnished titanium alloy Ti-6Al-4V. The yield strength of titanium alloy Ti-6Al-4V is commonly affected by the plastic deformation and two-phase distribution of material after ultrasonic burnishing. The plastic deformation degree of titanium alloy Ti-6Al-4V material has a greater impact on the yield strength.