Objective To prepare and evaluate Enterotoxigenic Escherichia coli（ETEC） reference.Methods The standard strain CMCC（B）43208 was identified by biochemical reaction， matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF MS） and gene sequencing. 600 ETEC reference samples with 10³ CFU/sample were prepared by freeze-drying. 20 samples were selected randomly for uniformity test， and samples were tested transportation stability by simulating 25 and 37 ℃， and storage stability by stroing samples at -20 and 4 ℃. Three laboratories were organized for collaborative calibration. 20 kinds food were chosen as the substrates to evaluate the applicability. The strain CMCC（B）43208 was identified as Escherichia coli by all of biochemical reaction， MALDI-TOF MS and 16 s RNA gene sequencing， and was identified as ETEC by lt，stp and sth gene. The uniformity result was F=1.48 <F_INV（0.05， 19， 20）by one-way anova. The samples were still 10³ CFU/sample after storing 7 d at 25 and 37 ℃， 28 d at 4 ℃ and 60 d at -20 ℃. All the results of three laboratories were ETEC （10³CFU/sample）.Results All 20 food substrates presented ETEC positively when adding the ETEC samples， while substrates presened ETEC negtively with no samples adding， the standard strain CMCC（B）43208 was ETEC.Conclusion The prepared samples were uniformit and stable， and the results of the collaborative calibration and applicability evaluation achieved expected requirements as 10³ CFU/sample， so the samples can be used as ETEC reference.