Antibiotic resistance in Escherichia coli is consistently highest for antimicrobial agents that have been in use the longest time in human and veterinary medicine. This study was carried out to detect the location of antibiotic-resistant gene in biofilm forming E. Coli isolates by molecular approaches. The investigated genes included CEF (E1) Cefidinir and AZT (E2) Aztreonam. According to biochemical experiment, E.coli isolates were recognized from sewage water. After that Antibiotic susceptibilities of E. coli isolates were tested against 14 antimicrobial agents by the disk diffusion method. The resistance of E. coli was found against cefidinir and Aztreonam. Biofilm forming ability of E. Coli was investigated by Microtitre plate assay, cover slip assay and tube assay. E. coli shows biofilm forming ability by all 3 methods. Further, we have done molecular characterization of resistant E. Coli. Detection of the 16s rRNA gene using universal primers was done. PCR technique was used to detect specific antibiotic resistance genes. The results indicate that biofilm forming resistant isolate EC1 (E. coli strain 1) contains Cefidinir resistance gene at 742bp on 100bp ladder marker and EC2 (E. coli strain 2) contains Aztreonam resistance gene at 478bp in the context of 100bp ladder marker. Both biofilm forming resistance isolates of E. coli harbour antibiotic resistance genes. The present study explores; in vitro qualitative estimation of biofilm production of E. Coli isolated from sewage and correlates it with antibiotic resistance.