The hot-section components are prone to fatigue failure due to severe working condition of high rotational speed and high cyclic load at high temperature. This study investigates the fatigue behavior of GH4169 Ni-based superalloy at 650 °C fabricated by laser directed energy deposition (L-DED) and proposes an improved high-temperature fatigue resistance by regulating the heat treatment. To this purpose, three conditions of solution and aging heat-treated fatigue specimens, namely 980STA, 980S5hDA, and 1050STA, were assessed. Results show that the fatigue resistance of the L-DEDed GH4169 with different heat treatments is mainly determined by the short crack propagation behavior, which is influenced by the combined effect of grain size and morphology. Finer grains with less texture strength of the 1050STA fatigue specimen has the longest short crack propagation life, while a cluster of large grains oriented to the 〈001〉 orientation of 980S5hDA leads to the most significant short crack propagation rate. Besides, the long fatigue crack propagation under the effect of strength and precipitates also minorly influence the fatigue resistance. The microstructure of interconnected precipitates including the needle-like δ phase and slender-stripe shaped Laves phase in 980STA and 1050STA specimens has a better hindering effect on the fatigue crack propagation than that of the discrete δ phase in 980S5hDA specimen, and the 1050STA specimen has the best strength. As a result, the 980S5hDA specimen has the worst fatigue performance, while the 1050STA specimen has the best. Compared to longer solution time, higher solution temperature can improve the fatigue resistance of the L-DEDed GH4169.