Introduction:Medical therapy has become increasingly difficult as drug-resistant Pseudomonas aeruginosa strains have emerged. The formation of biofilms and antibiotic resistance are two virulence mechanisms that contribute to the persistence of chronic illnesses. ExoU is a powerful Pseudomonas aeruginosa cytotoxin that is transported into host cells via the type III secretion system. Aim and Objective: To Study the Molecular characterization of ExoU Gene from Pseudomonas aeruginosa and its Antibiogram with the ability of Biofilm Formation in patients. Material & Methods: This was a crosssectional study carried out in the Department of Microbiology at a tertiary care centre for a period of 12 months 1.e, May 2023 to May 2024. A total of 130 Pseudomonas aeruginosa isolated from different clinical samples including urine, sputum, ear swab, wound swab, pus were identified by standard microbiological techniques. The isolates were further tested for MBL by Imipenem – EDTA combined disc test and MBL E-test (Imipenem). The DNA was isolated by Qiagen DNA extraction kit as per the manufacture’s guidelines and the resistant gene Exo U was detected by the conventional PCR. Results: A total of 400 clinical samples were studied out of which 130 P. aeruginosa isolates were obtained. The prevalence rate of Pseudomonas aeruginosa was observed to be 32.5%. It was observed that the Sensitivities of Colistin was (96.6%), Piperacillin‑tazobactam (77.6%), Amikacin (77.6 %), and cefepime (74.6 %) were found to be the most effective Antibiotics. The resistance to ciprofloxacin was (46.1%), Levofloxacin (53.8%), Gentamicin(62.3%), Imipenem (67.6%), Tobramycin( 68.4%), Ceftazidime (68.4%). There were 25(19.2%) were MBL positive by Imipenem – EDTA combined disc test, and 24(18.4%) by MBL E- test. In the present study it was observed that 67 (51.5%) of the Pseudomonas aeruginosa isolates showed biofilm formation. The molecular characterization confirms 25 (19.2%) expression exoU gene. Conclusion: In our study, the MBL E-Test was found to be the most effective phenotypic approach for detecting MBL. The recognised importance of the ExoU virulence gene in P. aeruginosa pathogenesis would benefit in the treatment and prognosis of Pseudomonas infections.