Aim & Objectives: The present study was aimed to determine phenotypic characterization of virulent Pseudomonas aeruginosa and its Type 3 Secretion system from Surat region. Methods: 125 clinical Pseudomonas aeruginosa were procured from different laboratories of Surat and 53 environment Pseudomonas aeruginosa isolated from 125 various environment and vegetable samples. Antimicrobial susceptibility testing was performed by Kirby Bauer Disc Diffusion method. The phenotypic screening evaluated for six soluble virulence factors (amylase, Caseinase, gelatinase, haemolysins, lecithinase, and lipase). Biofilm formation was detected by Tube method and Congo red agar method. Four virulence genes (exoS, exoT, exoU, exoY) were screened in selected isolates by PCR. Results: A high percentage of resistance rates (89.60% to amoxyclav and 88% to Cefuroxime) were observed amongst most of the isolates. Soluble enzymes were expressed in variable proportions (Gelatinase 96.80%, Lipase 96%, Lecithinase 88%, and Caseinase 88%). Tube method was considered to be superior to congo red agar method. The most frequent virulent gene detected was exoU (100%). Conclusions: Clinical and environmental virulent Pseudomonas aeruginosa harbour various genes that represent type 3 secretary systems and have high level of resistance that may result in to therapeutic failure.