Fatigue crack growth properties of materials are crucial for evaluating damage tolerance in additive manufacturing (AM) metallic structures. However, the unique microstructures and defects of AM materials result in highly complex fatigue crack growth behaviors. Currently, there is a lack of systematic fatigue crack growth rate measurement methods specifically targeting this characteristic. Therefore, taking directed energy deposited Ti-6Al-4V titanium alloy as the object of the study, fatigue crack growth tests were conducted in three orthogonal build orientations of the material using marker load method. Additionally, the visual measurement and compliance methods were also employed to measure fatigue crack growth rates, and the anisotropy of fatigue crack growth property was analyzed. Subsequently, anisotropic fatigue crack growth behaviors were characterized by optical microscope, scanning electron microscope, confocal microscope, and electron backscatter diffraction, suggesting that the microstructure is the primary factor affecting overall fatigue crack growth. Furthermore, nanoindentation tests were conducted to obtain the micromechanical properties within and among columnar grains in different build orientations, clarifying the homogeneity and anisotropy of mechanical properties. Finally, a fatigue crack growth rate measurement method based on marker load method was established, and the advantages of this method in AM materials and structures were summarized by comparing the results with those obtained using these two mature methods.