Abstract: To optimize the methods for concentration and DNA extraction of two pathogens in artificial lettuce infected with Enterohemorrhagic E.coli and Enteroinvasive E.coli. This study compared the concentration effect of different kinds of filtration membranes,and selected the best method of DNA extraction to extract the two athogens in lettuce sample. The multiplex PCR was used to detect the artificial lettuce infected with two pathogens. The results showed that the nylon membrane which had the pore size of 15 μm combined with mixed membrane which had the pore size of 0.22 μm was the best enrichment method. And the best DNA extraction method was slected by using the kit of bacterial genomic DNA extraction. The detection limit of lettuce samples by using multiplex PCR was decreased 10 times. The detection time was saved 1.5 h. The multiplex PCR combined with the optimized method was sensitive,fast and easy. This study had a high practical value.