In this study, high-strength steel FV520B sheets were subjected to high-cycle tensile-tension fatigue experiments at room temperature in order to obtain fatigue-damaged specimens. Then Lamb waves were used to perform nonlinear ultrasonic testing on them to obtain the normalized relative nonlinear coefficients, β′/ β0. The corresponding relationship between β′/ β0 and the percentage of fatigue life was obtained. Finally, the microstructural changes of the damaged samples were observed by a scanning electron microscope in order to explore the correlation mechanism between β′/ β0 and the degree of micro-defects and fatigue damage. The experimental results showed that as the number of fatigue cycles increased, β′/ β0 first slowly rose, then quickly rose to reach a peak, and finally declined, which was consistent with the generation and propagation of dislocations and cracks during fatigue damage. As the length and number of microcracks increased, β′/ β0 also increased, especially in the initial stage of fatigue crack initiation. β′/ β0 was very sensitive to the size of fatigue cracks, so the change of β′/ β0 can be used to detect the degree of early fatigue damage of the material.