Staphylococcus aureus (S. aureus) is a common pathogen that is dangerous to humans' health. Herein, a novel upconversion fluorescent biosensor based on fluorescence resonance energy transfer from aptamer-labeled upconversion nanoparticles (UCNPs-apt) as donor and cobalt oxyhydroxide (CoOOH) nanosheets as acceptor was designed to detect S. aureus in complex matrices. The principle of the work relies on fluorescence resonance energy transfer as UCNPs-apt can self-assemble on CoOOH nanosheet surfaces by van der Waals forces to effectively quench the fluorescence. When S. aureus was added, the aptamer was able to preferentially capture the target, resulting in the dissociation of donor and acceptor and the recovery of fluorescence. The structure and morphology of the nanostructures were assigned in detail by a series of characterizations, and the energy transfer mechanism was evaluated by time-resolved lifetime measurements. Under the optimal conditions, a linear calibration plot was obtained in a concentration range of 45–4.5 × 106 CFU/mL with a limit of detection of 15 CFU/mL. In addition, the proposed biosensor was used for S. aureus detection in real samples (e.g., pork, beef), and the detection result showed no significant difference (p > 0.05) compared with the conventional plate count approach. Hence, the fabricated biosensor holds a potential application for S. aureus in food analysis and public health. [ABSTRACT FROM AUTHOR]