Staphylococcus　aureus　(S.　aureus)　is　a　prevalent　foodborne　pathogen　that　could　cause　severe　food　poisoning.　Thus,　rapid,　efficient,　and　ultrasensitive　detection　of　S.　aureus　in　food　samples　is　urgently　needed.　Here,　we　report　an　efficient　magnetic　enrichment　cascade　single-step　recombinase　polymerase　amplification　(RPA)-CRISPR/Cas12a　assay　for　the　ultrasensitive　detection　of　S.　aureus.　Magnetic　beads　(MBs)　functionalized　with　S.　aureus-specific　antibodies　were　initially　used　for　S.　aureus　enrichment　from　the　complex　matrix,　with　98%　capture　efficiency　in　5　min　and　100-fold　sensitivity　improvement　compared　with　unenriched　S.　aureus.　Next,　a　single-step　RPA-CRISPR/Cas12a-based　diagnostic　system　with　optimized　extraction-free　bacteria　lysis　was　constructed.　This　assay　could　detect　as　low　as　1　copy/μL　(five　copies/reaction)　of　extracted　DNA　template　and　10　CFU/mL　of　S.　aureus　within　40　min.　Furthermore,　the　assay　could　effectively　detect　S.　aureus　in　real　food　samples　such　as　lake　water,　orange　juice,　pork,　and　lettuce,　with　concordant　results　to　qPCR　assays.　The　proposed　cascade　signal-amplification　assay　eliminates　the　need　for　lengthy　bacterial　culture　and　complex　sample　preparation　steps.　Hence,　the　proposed　assay　shows　great　application　potential　for　rapid,　efficient,　and　ultrasensitive　detection　of　pathogens　in　real　food　samples.　©　2024　Elsevier　B.V.