To develop a fast and sensitive Real-Time PCR method with a short (3-5 h) enrichment to detect and quantify E.coli in food.Methods Experiments were performed with E.coli(ATCC 25922) as the reference strain. The optimal enrichment conditions were established from different medium and different temperature. Both quantification calibrators and samples were enriched for 3-5 h in the optimal enrichment medium. DNA was extracted using Triton-X 100 method, and was amplified using an E.coli-specific PCR assay targeting the specific gene. Standard curves were created by plotting the cycle threshold (Ct) values against the logarithm of the original quantity of reference strain (before enrichment), and samples were calculated from the respective Ct value.Results Under the conditions of pure E.coli growing in NB, standard curves showed a good linearity, with r²of 0.996,0.992 and 0.991 for 3,4 and 5 h enrichment respectively, and the corresponding limits of detection (LOD) were 136,4 and 1.4 cfu/100 ml, respectively. This method was validated by creating standard curves after the 4 h enrichment in NB and EC with background microorganisms at 42.0 ℃,with r²of 0.972 and 0.978, respectively. This method also tested the natural and spiked food samples, with a recovery rate from 74.0% to 174.0%. Conclusion The Real-Time PCR with 3-5 h enrichment is a rapid, accurate and reliable technique for quantifying the viable E.coli cells in food..