Staphylococcus aureus (S. aureus) is a widely prevalent Gram-positive bacteria that can cause serious infections and diseases in humans and other organisms. Timely detection and treatment in clinical settings is crucial for patient safety and public health. However, current methods for S. aureus detection still face some limitations, such as time-consuming operation, false positives, and labor-intensive available methodology with low sensitivity. Therefore, it is particularly important to develop a rapid, simple, sensitive, and cost-effective method for detecting S. aureus. We developed a SERS platform based on allosteric aptamer-triggered catalytic hairpin assembly (CHA) and photoactivated CRISPR/Cas12a reactions, combined with a multifunctional core-shell structure as the SERS reporter, enabling highly sensitive one-pot determination of S. aureus. Compared with traditional two-step and one-pot analysis methods, this strategy offers superior sensitivity and can successfully identify real samples contaminated with S. aureus. The platform utilizes light-controlled CHA and CRISPR/Cas12a reactions, effectively preventing interference between different reaction systems. Therefore, the photoactivated one-pot CHA/Cas12a strategy provides a simple, rapid, highly sensitive, specific, and cost-effective method for one-pot determination of S. aureus in clinical samples.