Introduction: Biofilm are surface associated bacterial communities surrounded by a matrix of exo-polymers and are responsible for development of clinical infections.Urinary tract infections are considered to be one of the most common bacterial infections.Uropathogenic strains of Escherichia coli account for 70-95% of the UTIs. The bacteria enclosed within the biofilm are extremely resistant to treatment. Objective: To study Biofilm formation of Uropathogenic Escherichia coli by Tube method and Tissue Culture Plate method. Material and Methods: The study was carried out at Department of Microbiology, MGM Medical College, Kamothe, Navi Mumbai from October 2015 – September 2016. Total 200 urine samples were processed. Out of 200 samples, 100 isolates of Uropathogenic Escherichia coli were included in this study. They were identified by standard microbiological procedures. These isolates were subjected to biofilm production by Tube method and Tissue culture plate method. Results: Out of 102 Uropathogenic Escherichia coli isolates 40.19% were biofilm producers by Tube Method and 47.05% by Tissue culture Plate Method. Conclusion: Tube Method correlated well with Tissue Culture Plate method for strong biofilm detection in Uropathogenic Escherichia coli.