We have experimentally investigated the cyclic deformation in copper using ultrasonic nonlinearity. The observation and characterization of dislocation substructure have been conducted using transmission electron microscope and electron backscattered diffraction technique. The ultrasonic nonlinearity (${eta}/{eta}_0$) was measured by the harmonic generation technique after various fatigue cycles. The microstructural effect on the nonlinearity was discussed regarding the extent of dislocation substructures evolved from low cycle fatigue. The ultrasonic nonlinearity of copper monotonically increased with the fatigue cycles due to the evolution of dislocation cell substructures.