The burst-mode femtosecond laser has the potential to be a novel thrombus removal technique. This paper proposed to investigate the mechanism of thrombus ablation in vitro by burst-mode femtosecond laser. A simulation model of the interaction between femtosecond laser and thrombus was established. An in vitro thrombus model was prepared. Combined with the high-speed galvanometer and femtosecond laser, the ablation experiments in vitro were performed. The experimental results showed that the ablative threshold was 0.27 times and the efficiency was about 1.4 times of burst-mode femtosecond laser as those of traditional mode femtosecond laser. These phenomena were related to the residual temperature and free electrons on the thrombus surface, which confirmed the simulating results and had relationship with incubation effects. The high ablative efficiency and safety of burst-mode femtosecond laser for thrombus ablation were verified, which may help to achieve the femtosecond pulse output through flexible fiber easily and stably. The burst-mode femtosecond laser represents an important technological advancement of the method in terms of endovascular treatment with femtosecond laser.