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Healthcare-associated infections (HAIs) have increasingly become a threat to global health. Rapid, accurate, and reliable detection of HAI-related bacteria is urgently needed. Here, we presented a novel antimicrobial peptide-based surface-enhanced Raman scattering lateral flow assay (SERS-LFA) for the detection of multiple HAI-related bacteria. Antimicrobial peptide cecropin 1(CP1) was immobilized on the surface of Fe 3 O 4 @Au by the streptavidin-biotin system to capture Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli O157:H7 (E. coli O157:H7) simultaneously. The synthesized Fe 3 O 4 @Au-CP1 exhibits excellent broad-spectrum capturing ability, rapid magnetic enrichment function, and superior antibacterial activity. LFA strips enable the specific detection of two target bacteria in a quick and simple manner, with bacterial type differentiation via corresponding monoclonal antibodies sprayed. The highly sensitive SERS signals and the amplification effect of streptavidin-biotin significantly improved the sensitivity of detection. The LODs of P. aeruginosa and E. coli O157:H7 were 12 and 16 cells/mL, respectively. This assay also exhibited good specificity and reproducibility when applied to urine samples. Therefore, Fe 3 O 4 @Au-CP1-based SERS-LFA has great potential for detecting multiple bacteria after infection as well as regularly monitoring therapeutic efficacy. • CP1 was first utilized as a broad-spectrum recognition element in SERS-LFA to detect multiple bacteria simultaneously. • The amplification effect of the streptavidin-biotin system enhances the binding efficiency of Fe 3 O 4 @Au-CP1 to bacteria. • Fe 3 O 4 @Au-CP1 MNPs enable quick capture and enrichment of bacteria. • CP1-based SERS-LFA can achieve highly sensitive and quantitative detection for P. aeruginosa and E. coli O157:H7. • The LODs for P. aeruginosa and E. coli O157:H7 were 12 cells/mL and 16 cells/mL, respectively. [ABSTRACT FROM AUTHOR]