In order to study the characteristics of streamer discharge in sub-millimeter gap, an experimental system and an image acquisition system of positive and negative polarity streamer discharge in the gap of 100–500 μm at atmospheric pressure are built, and the initial breakdown voltages and breakdown images of positive and negative polarity streamer discharge in a gap range of 100–500 μm are collected, and the fractal dimension of the experimental discharge images is calculated by Matlab. The experimental analysis shows that the air breakdown discharge in the gap range of 100–500 μm still conforms to the stream theory, and the polarity effect between the breakdown voltage of positive and negative streamers and the fractal dimension of the discharge image is obvious. The influence of space charge and photoionization on the meandering degree and coverage of the discharge channel in stream theory increase with the increase of the gap, and jump at 300 μm. The fractal simulation of positive and negative streamer discharge with a gap of 500 μm is established by using WZ (Wiesmann-Zeller) model based on fractal theory in Matlab. It is found that the development probability index η is inversely proportional to the fractal dimension, and that the η is smaller in positive streamer than in negative streamer under the same discharge condition.