Abstract: E. coli is the major bacterial pathogen which can cause outbreaks of food borne illness and it is also an indicator of different fecal contamination. The present study was aimed to isolation and identification of the E. coli in the samples of refrigerated chicken rinse obtained from different super stores under Chittagong City Corporation, Bangladesh. Packages of chicken meat were opened aseptically strictly and total 1000 mL chicken rinse were transferred to 100 mL of PBS and incubated at 37°C for 24 h. Aliquots were transferred to Nutrient Broth medium and incubated at 37°C for overnight. After incubation the aliquots were streaked in MacConkey Agar media and incubated at 37°C for overnight. Typical E. coli colonies were picked from selective agar media and restreaked on EMB (Eosine Methylene Blue) medium. Several microbiological and biochemical tests were performed to confirm the suspected E. coli. Moreover, a PCR was developed to detect the microorganism in the chicken rinse by molecular method. The total DNA obtained by thermal extraction of microorganism was amplified using two set of primers designed for specific region of E. coli 23S rRNA gene and presence of amplicon 662 bp was found correlated to the result obtained from the standard culture method. All the samples were resistant to two or more than two antibiotics. The sample showed 100% resistant to Doxycycline and Oxytetracycline. But they were uniformly sensitive to Ampicilin. 100% susceptibility was found for Gentamycin group. The second susceptibility result was found in case of Chloramphenicol group. The prevalence of E. coli infection in frozen chicken rinse was found 77.51 and 100% by bacteriological and molecular techniques respectively in this study.