Objective A method for the preparation and certification of the reference material of tenuazonic acid(TeA) and tentoxin (TEN) in wheat flour was developed. Methods The reference material of TeA and TEN in wheat flour was prepared from grain which naturally contaminated alternaria toxins, characterizated with national standard regulation. Results The reference materials of TeA and TEN in wheat flour which contains two values were developed. The reference materials were approved the second class of National Certified Reference Materials [GBW(E) 100547 and GBW(E) 100548]. Conclusion The reference materials are currently the only National Certified Reference Materials of wheat flour that naturally contaminate TeA and TEN. The reference materials provides important method ological reference for the development of matrix reference materials with new mycotoxins in grain in China.