In order to obtain the standard substance 3-nitro-1,2,4-triazol-5-one (NTO), it is necessary to establish the corresponding chemical certification method in its preparation. Using methanol as solvent and sodium methanol standard solution as titrant, the certification of NTO standard substance was investigated by non-aqueous titration. The results show that the method can exclude disturbance of impurity with better accuracy and precision, and the average purity value of NTO standard substance is 99.88% and the standard deviation is 0.16% after 27 times of repeated determinations.