The sterilization effects of surface E. coli bacteria by N2/O2 source atmospheric pressure plasma jet (APPJ) were studied, and the spectroscopic properties of nitrogen/oxygen source atmospheric pressure plasma jet were analyzed. Experimental results showed that under discharge voltage at 6.8 kV, gas flow rate at 4L?min?1 and treatment time at 3min, E. coli bacteria inactivation rate of APPJ with N2/O2 at 1:4 reached 98.4%, close to that of oxygen source APPJ. Active substances of nitrogen/oxygen source plasma jet were analyzed by optical emission spectroscopy (OES) to interpret the mechanism of the microorganism of E. coli bacteria, and it was considered that active substances, such as NO?γ, OI, ?OH, played the important roles in the surface E. coli bacteria inactivation process. This would provide the experimental basis and technical support of application researches of atmospheric pressure N2/O2 plasma jet, such as environmental hygiene, microbial inactivation and so on.