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Fatigue damage and micro-cracks growth have been proved to be closely related to the nonlinear effect of ultrasonic Lamb waves propagation in materials. In this paper, the relationship among fatigue life, length and width of micro-cracks, and the acoustic nonlinearity parameter was established. The variation of the acoustic nonlinearity parameter caused by fatigue damage was analyzed based on micro-cracks growth. Considering the multimodal nature of nonlinear Lamb waves, we selected the low-frequency mode pairs S0–s0 and S1–s2 to evaluate fatigue damage in 7075 aluminum alloy plates. Both FE simulations and experiments show that the mode pair S1–s2 is more suitable than S0–s0 for the assessment of fatigue damage. It was found that with increasing fatigue cycles, the relative acoustic nonlinearity parameter A2/A12 firstly increases because the micro-cracks length extends while the micro-cracks width does not change much. The relative acoustic nonlinearity parameter A2/A12 reaches its peak at about 60% of the fatigue life. Then the relative acoustic nonlinearity parameter A2/A12 decreases because of the rapid increase in the micro-cracks width. Our findings clarify the mountain-shape curve between the relative acoustic nonlinearity parameter A2/A12 and the fatigue cycles. [ABSTRACT FROM AUTHOR]