A method of testing colibacillus O157 with MGB probe multiline fluorescence gauging PCR, is disclosed, the recombinant plasmid is constructed, the synthesized primer and the Taqman-MGB probe are designed, diploid fluorescence gauges PCR, the segment of saving sequences of rfbE, vt2 of the colibacillus O157 are augmented by PCR to obtain the goal segment, the goal segment is connected with PMD 18-T Vector to obtain the recombinant plasmid, then the recombinant plasmid is converted to DH5a competence bacillus coli cell, enlarged the culture to obtain the recombinant plasmid with high concentration, then the recombinant plasmid with high concentration is diluted by 10 times ratio to be the standard sample, the standard sample and the sample to be measured are combined together to gauge the PCR by diploid fluorescence, the calibration curve is formed by the standard sample, the content of the colibacillus O157 can be calculated in the sample to be measured based on the calibration curve. The MGB probe is used in the invention that has higher sensibility and specificity, and detects the content of the colibacillus O157 in the sample quickly and precisely; two specificity virogenes of the colibacillus O157 can be detected at the same time, so it is a quick and precise method of testing colibacillus O157.