Nitrogen isotope separation using plasma chemical reactions in N₂–O₂ glow discharge has been investigated with a DC power supply being used to generate the glow discharge. In this study, the effects of parameters such as oxygen concentration, discharge pressure and discharge current on this process are studied experimentally. We determined that the separation factor correlates strongly with the discharge current, of which the optimum value is 0.4 mA for the highest separation factor of β = 1.57. The conversion of nitrogen increases with increases in the discharge current and oxygen concentration. The conversion of oxygen is found to have a value of nearly 100% and almost all of the oxygen input was converted into ozone molecules. The electron density also is estimated and its influence on the isotope separation is discussed. The results of this experimental study correlate with those of the theoretical study in literature concerning the relationship between electron density and separation factor.