Objective To satisfy the demands of laboratory quality control and proficiency testing for the detection of enteroinvasive Escherichia coli (EIEC), a stable EIEC reference material was developed with independent intellectual property rights, good stability and clear background genomic information. Methods NGS Technique was used to sequence the whole genome of enteroinvasive Escherichia coli (CMCC 44840), bioinformatics analysis was performed to identify the species, serotype, MLST type and virulence genes; EIEC lyophilized samples with 10³ CFU were prepared by freeze-drying method ; The homogeneity test was performed according to CNAS-GL017, and the result were statistically analyzed by one-way ANOVA; The samples were stored at -20,4, 25 and 37 ℃, respectively, and their storage stability and transport stability were evaluated; Five kinds of food matrix samples were used to verify the applicability of reference materials, and three laboratories were organized for collaborative calibration. Results The genome size of CMCC 44840 was 4.96 Mb, the GC content was 50.7%, and the genome contained 5424 coding genes. This strain was identified as the species of Escherichia coli, and the serotype and the MLST type were predicted to be O28ac:H7 and ST311, respectively, and it carried ipaH, virB, virF and other virulence genes. The homogeneity test result of the EIEC lyophilized sample was F=1.79, which met the homogeneity requirements of reference materials. The EIEC samples were still 10³ CFU when storing at -20 ℃ and 4 ℃ for 60 d and storing at 25 ℃ and 4 ℃ for 7 d, and decreased on the third day at 37 ℃. EIEC were detected in 20 different food matrix samples with EIEC lyophilized samples after enrichment. The samples were 10³ CFU determined by three collaborative calibration laboratories, and there were no significant differences among these laboratories (F=0.59). Conclusion The EIEC reference material prepared in this study has clear genome sequence information. The homogeneity and stability meet the requirements and have good applicability, which can meet the requirements of quality control and capability verification in food testing laboratory.