In this paper, the method of experimental research is used to minimize the micro-vibration amplitude of 0.6 mm thick glass substrates in the air conveyor system. The optimal parameter combination of the small orifice throttling glass substrate air conveyor system is obtained as follows: orifice diameter 0.25 mm, orifice spacing 60 mm and air-film thickness 50 μm. Secondly, based on the orthogonal experimental data, the influence of the main parameters of the air conveyor system at the micro vibration of the glass substrate is analyzed by variance analysis technology. The results show that the three principal parameters of the air conveyor system have significant influence on the vibration of the glass substrate. Among them, the thickness of the air-film has the greatest influence on the vibration of the glass substrate, the diameter of the orifice has the second influence, and the spacing of the orifice has the least influence. Finally, the influence of the three key parameters of orifice diameter, orifice spacing and film thickness on the micro vibration of glass substrate in the orifice throttling air flotation support system was studied and discussed, and the relationship curves between the maximum amplitude of micro-vibration of glass substrate and the main parameters of air flotation system were obtained.