Detection of pathogenic bacteria in complex biological matrices remains a major challenge. Herein, we report the selection and optimization of a new DNAzyme for
Staphylococcus aureus(SA) and the use of the DNAzyme to develop a simple lateral flow device (LFD) for detection of SA in nasal mucus. The DNAzyme was generated by in vitro selection using a crude extra/intracellular mixture derived from SA, which could be used directly for simple solution or paper‐based fluorescence assays for SA. The DNAzyme was further modified to produce a DNA cleavage fragment that acted as a bridging element to bind DNA‐modified gold nanoparticles to the test line of a LFD, producing a simple colorimetric dipstick test. The LFD was evaluated with nasal mucus samples spiked with SA, and demonstrated that SA detection was possible in minutes with minimal sample processing.