Background: Staphylococcus aureus (S. aureus) a pathogenic bacterium typically found in the population is noteworthy for its pathogenicity due to its ability to produce biofilms with high virulence. Aِim: To identify the gene icaA, which is responsible for biofilm formation in clinical isolates from wounds and urine samples. Methods: This gene is involved in the construction of slime layers. The present study collected a total of 425 clinical samples, including urine and wound swabs. We used four different methods to identify S. aureus isolates: the biochemical test methodology, the Analytical Profile Index (API) Staph system, the Vitek-2 compact systems, and the polymerase chain reaction (PCR) targeting the 16SrRNA. In the end, we confirmed only 388 isolates to be S. aureus. Results: We assessed the ability of S. aureus isolates to produce biofilm using 96-well microtiter plates. The examination of 388 isolates revealed that 146 isolates (37.6%) formed robust biofilms, 160 isolates (41.3%) formed moderate biofilms, and 82 isolates (21.1%) formed weak biofilms. Additionally, the expression level of the strong biofilm-producing isolate (6.508) was considerably lower (P<0.01) compared to the weak and moderate isolates. The results indicated that the moderate isolate's expression level was higher than that of the weak isolate. Conclusion: A quantitative PCR analysis was conducted on three isolates with different biofilm forming capacity (EPS) to evaluate the transcript levels of the icaA gene, which is responsible for exopolysaccharide production.