Nickel-based single-crystal superalloys are widely used in high-temperature turbine components, where fatigue resistance is critical to service reliability. Low-angle grain boundaries (LAGBs) are inevitably formed in directionally solidified nickel-based single-crystal superalloys, and their misorientation plays an important role in governing high-temperature fatigue performance. In this study, DD413 nickel-based single-crystal superalloy bicrystal specimens containing LAGBs with misorientation angles of 4.8°, 10.8°, and 13.3° were prepared, and their high-temperature fatigue behavior was systematically investigated at 900 °C. The results show that fatigue life decreases significantly with increasing misorientation, and the fatigue life decreases more markedly when the misorientation increases from 10.8° to 13.3°, indicating aggravated grain-boundary-related damage in the higher-misorientation range. At a maximum stress of 600 MPa, the fatigue life of the specimen with a misorientation of 13.3° is approximately 5% of the 4.8° specimen. With increasing misorientation, the fatigue fracture mode gradually transitions from matrix-dominated cracking to grain-boundary-dominated cracking. Correspondingly, pronounced strain localization develops in grain-boundary-adjacent regions, accompanied by enhanced elemental segregation characterized by Cr enrichment and Al depletion, as well as increased atomic-scale structural instability. Multiscale microstructural characterizations combined with molecular dynamics simulations demonstrate that the misorientation of LAGBs is a key structural parameter governing interfacial stability and defect accumulation during high-temperature cyclic loading. These findings provide a mechanistic basis for misorientation tolerance control and defect management in single-crystal turbine blade applications.