The availability of nutrients is closely linked to the protein expression profile of microorganisms. In this study, a comparative investigation has been performed to examine the expression patterns of cell associated proteins and secreted proteins in Staphylococcus aureus cultivated in TSB and milk media at three time points: 3 h, 9 h, and 18 h of the growth stage using proteomic techniques. In TSB medium, cell-associated proteins such as acetyl-CoA acyltransferase, acyl-CoA synthetase, argininosuccinate lyase, and argininosuccinate synthase were detected at higher levels at 9 h and 18 h compared with 3 h, indicating overall increases during growth. While in milk, quinone oxidoreductase 1 and zinc-type alcohol dehydrogenase-like protein SAR2277 exhibited a significant increase across 3, 9 and 18 h. Secretory proteins such as δ-hemolysin, phospholipase C and panton-valentine leukocidin F showed significant increases in both media across 3, 9 and 18 h. Notably, cell associated proteins or secreted proteins such as the leukocidin S subunit and staphopain B were significantly upregulated in TSB medium, while thermonuclease and transcriptional regulator SarA were found to increase in milk. These findings highlight the importance of the culture medium in determining the relative abundances of cell associated proteins and secretory proteins in S. aureus , providing insight into microbial adaptation mechanisms. Furthermore, these up-regulated proteins may serve as potential biomarkers and have practical implications for improving control strategies, leading targeted approaches to reducing S. aureus contamination in milk and dairy products, and eventually contributing to better food safety and quality.