<p><i>Staphylococcus aureus</i> (<i>S. aureus</i>) is one of the leading pathogens responsible for bacterial food poisoning. To avoid <i>S. aureus</i> infections, it is essential to conduct rapid and accurate testing of food products. In this study, an aptamer-mediated CRISPR/Cas signal amplification system based on dendritic hybridization chain reactions (termed as Apt-dHCR-CRISPR/Cas12a) was constructed for high sensitive detection of <i>S. aureus</i> in food. Specifically, the biotin-modified linker was first enclosed by the <i>S. aureus</i> aptamer and attached to the surface of the magnetic beads (MBs) via biotin-streptavidin interactions. Hybridization chain reaction was then used to construct dendritic DNA nanomolecules that provided multiple binding sites for Cas12a-crRNA. Once encountering <i>S. aureus</i>, the aptamer underwent an adaptive conformational change, creating a unique binding site that enabled it to bind specifically with <i>S. aureus</i>, thereby exposing the linker. The dendritic DNA nanomolecules then bound to the exposed linker, anchoring onto MBs for retention. Finally, Cas12a activation via crRNA-target engagement triggered collateral cleavage of fluorescent probes, enabling ultrasensitive fluorescence signal amplification. Benefiting from the remarkable signal amplification property of dendritic HCR and the specific recognition ability of Cas12a, the proposed Apt-dHCR-CRISPR/Cas12a system exhibited excellent detection sensitivity and specificity, achieving the LOD as low as 18&#xa0;CFU/mL with a linear calibration range of 10² to 10⁷&#xa0;CFU/mL, demonstrating good discrimination of different bacterial species. In addition, it was further successfully applied to milk samples, demonstrating that this Apt-dHCR-CRISPR/Cas12a system has great potential to improve the accuracy of screening for food contamination.</p>