The invention provides a quadruple fluorescent quantitative polymerase chain reaction (PCR) detection kit for diarrheagenic Escherichia coli. The quadruple fluorescent quantitative PCR detection kit consists of fluorescent quantitative PCR reaction liquid, a standard substance O157, a standard substance H7, a standard substance O104, a standard substance H4 and a reference substance, wherein the fluorescent quantitative PCR reaction liquid comprises PCR reaction buffer solution (which contains a mixture of magnesium chloride and deoxyribose nucleotide triphosphate, heat-resisting thermus aquaticus deoxyribonucleic acid (Taq DNA) polymerase and the like), special upstream and downstream primers and a probe. In the quadruple fluorescent quantitative PCR detection kit, O157:H7 and O 104:H4 type bacteria of the diarrheagenic Escherichia coli can be detected from a sample; and compared with the conventional ordinary PCR method and single fluorescent quantitative PCR method, the quadruple fluorescent quantitative PCR detection kit has the advantages of simplicity, convenience, quickness and accuracy; and the detected bacteria are quantitated accurately in real time, and the diagnosis of suspected patients infected with the bacteria clinically is clarified early, so that the treatment scheme is formulated in time to lower the death rate.