A control method of a brain computer interface (BCI) with a stepping delay flickering sequence is provided. First, a plurality of different flickering sequence signals are generated by encoding a static flashing segment and a plurality of stepping delay flashing segments divided in different time sequences. Then, a plurality of target images corresponding to the flickering sequence signals are displayed. Thereafter, a response signal generated by an organism evoked by the target images is acquired. Then, signal processing is performed on the response signal by using a mathematic method to distinguish which one of the target images is gazed by the organism. Thereafter, a controlling command corresponding to one of the target images is generated. A control system of a BCI with a stepping delay flickering sequence is also provided herein.