The objectives of this research was to establish the method of detecting E.coli DNA using real-time PCR assays and by which to detect the E.coli load in clinical blood samples primarily.Theβ-D-galactosidase gene of E.coli was selected as the target gene for designing primers and probe, the recombinant plasmid was constructed by standard template. Twenty-six samples of surgical febrile patients include blood and other body fluid samples. Results showed that the specificity of primers and probe was excellent, the average precision of the RQ-PCR equipment for various samples was 112.19%±7.48%; the intra- and inter-assay coefficients of variation were 16.33 %±4.8 %、16.03 %±7.8 %, respectively. The positive detecting limit was determined artificially to be 16 copies/well through forty healthy volunteers' venous whole blood samples were studied. Seventy-two blood samples of 31 febrile patients were collected to be studied by culture and real-time PCR assays. The positive rate of culture was 2.78%(2/72),while the positive rate of E.coli DNA in real-time PCR was 51.35%(38/74), and the difference was statistically significant(P＜0.01). The correlation between the quantity of E.coli DNA and body temperature, heart rate was significant(P＜0.01),and the degree of association was medium(r=0.57 and 0.43,respectively),and it were not significant between quantity of E.coli DNA and WBC count, percentage of neutrocyte and lymphocyte(P＞0.05).It is suggested that the real-time quantitative PCR is rapid, sensitive, specific ,less contamination and reproducible in detection of E.coli DNA. It can be used primarily in quantitative detection of E.coli DNA in various clinical specimens of body fluid. The positive rate of real-time PCR assays was much higher than culture-based methods. It can reflect the severity of E.coli bacteremia rapidly and exactly and then facilitate us to acquire the more precise, rapid, sensitive and effective evaluation of dynamic process or outcome of bacterial translocation induced by injury of gut barrier.