Hundreds of film-cooling holes with a diameter ranging from 0.2 mm to 0.8 mm are usually arranged on nickel-based single-crystal turbine blades. The existence of these film-cooling holes impairs the structural integrity of the turbine blades, making them high-risk locations for blade failure and fracture under high-temperature fatigue loads. To address the issue that the initial manufacturing defects of film-cooling holes are strongly correlated with their fatigue fracture behavior, a study on the fatigue fracture behavior was conducted using nickel-based single-crystal alloy specimens with film-cooling holes fabricated by femtosecond laser. Firstly, a three-dimensional spiral hole-drilling simulation model for femtosecond laser was established based on the hydrodynamic method to obtain the evolution law of hole morphology during the drilling process. Subsequently, the surface integrity of the film-cooling holes was analyzed from three aspects: geometry, microstructure, and mechanical properties. The average taper of the film-cooling holes was approximately 0.19°, the maximum depth of the thermal damage zone near the hole edges was about 18 μm with severe oxidation observed, and the maximum residual stress at the hole edges reached 463 MPa. Subsequently, fatigue tests were conducted on the specimens with film-cooling holes at 980 °C. Fracture analysis revealed that cracks originated from the heat-affected zone of the hole walls and propagated in an elliptical contour with an approximate 6° counterclockwise rotation until fracture occurred. Finally, based on the fatigue fracture behavior of film-cooling holes fabricated by femtosecond laser, an M-integral crack propagation driving force was proposed, which is based on incremental plasticity and configurational force theory. On this basis, an effective functional model describing the relation-ship was constructed between the fatigue crack growth rate of film-cooling holes and the effective crack propagation driving force.