Urinary tract infections (UTIs) are the most common type of illness in both elaborate and refined the world. Anyway the infections are more familiar in men, women and children; they are at a significant rate in men and of all age groups. A distinctive dare for clinical culture and infection control regular is to deal with extended-spectrum beta-lactamase (ESBL), which now frequently causes CAI, including UTIs, and indicate a challenge for exponent in choosing actual antibiotics. This study aimed to isolate ESBL-yielding bacteria from UTI, patients and analyse their phenotypic attribution. Materials and Method: For the study total numbers of 123 urine samples from UTI patients were collected from Kerala's different Medical College Hospitals; between year 2021- 2022. The isolates and identification of positive bacteria were retrieved and screened for ESBL production by gram staining, biofilm, and Double Disk Diffusion Synergy Test (DDST). Isolates with ESBL phenotype were further characterized by antibiotic resistance testing, sequencing, and phylogenetic trees of ESBL genes. Results: Total number of UTI patient urine sample for sixty three positive isolates were screened as E. coli on EMB agar plates and an IMViC biochemical test was conducted. MAR was determined using twelve different antibiotics using microtiter plate assay. Biofilm formation, weak, moderate, strong. The isolates tested, zone of inhibition ceftazidime, cefotaxime, ceftriaxone, were observed for isolates E. coli which showed resistance to third-generation cephalosporins were subjected to phenotypic confirmatory test by modified DDST to detect the ESBL production. Based on the confirmatory test, eight of the isolates were confirmed as ESBL producers. E.coli strain 16S rDNA U65 sample yield amplification products of approximately 1499bp.