The effects of the laser pulse duration and the number of pulses on the ablation threshold of Ni-based superalloy were experimentally investigated. In this experiment, Ni-based superalloy samples were irradiated by 1, 10, 50, 100, 300, 500 and 1000 laser pulses with different pulse duration, respectively, and pulse duration 290 fs, 1 ps and 7 ps were explored. The diameter of ablation holes expands with the increase of laser pulse and the effect of pulse number on the diameter of ablation holes increases with the pulse duration from the experiments. By analyzing the relationship between the diameter of ablation holes and laser pulse energy, multi-pulse ablation threshold of Ni-based superalloy irradiated by ultrafast laser with different pulse duration can be obtained. No matter what the pulse duration of the laser was, the incubation effects of multi-pulse irradiation on the surface of the superalloy were significant. And the incubation coefficient for the thresholds calculated by diameter respectively in 290 fs, 1 ps, and 7 ps is fitted to be 0.88, 0.86 and 0.78.