The interrupted fatigue test method was utilized to investigate the damage evolution mechanism of the notch high-cycle fatigue (NHCF) in Ti-55531 alloy with a multilevel lamellar microstructure. The results reveal that significant microvoids and microcracks predominantly initiate at α/β interfaces under various notch root radii (R). Notably, even under larger R (0.75 mm), mutual interactions of stacking faults (SFs)−deformation twins, twins−twins, and SFs−SFs are observed. Furthermore, with decreasing R (0.34 and 0.14 mm), the volume fraction of SFs escalates significantly and twins are almost absent. Moreover, activated prismatic slip system decreases with a decrease in Schmidt factor and with the further decrease in R. Finally, strain localization near α/β interfaces contributes to the initiation of fatigue microcracks.