<p>Excessive tryptamine in food poses a significant risk to human health, emphasizing the demand for efficient, sensitive, and rapid detection technologies. Surface-enhanced Raman Spectroscopy (SERS) aptasensors are presently attracting a lot of attention due to their ability to detect targets at low concentrations. However, their inherent instability and poor anti-interference ability hinder their widespread use. Herein, a two-layer core-satellite magnetic SERS aptasensor was constructed to achieve the sensitive detection of tryptamine. The SERS aptasensor consisted of magnetic SERS recognition probes (magnetite nanoparticles coated with gold nanoparticles and aptamer: Fe₃O₄@Au-apt) and SERS signal probes (gold nanoparticles of two different sizes functionalized with the Raman reporter molecule 4-mercaptobenzonitrile and complimentary deoxyribonucleic acid strands). In the absence of tryptamine, the SERS signal probes attached to the aptamer on the SERS recognition probes to form a two-layer core-satellite structure with an intense SERS signal at 2226&#xa0;cm^− 1 in the “biological-silent” region due to the 4-Mercaptobenzonitrile. In the presence of tryptamine, tryptamine bonded to aptamer on the SERS recognition probe leading to detachment of the SERS signal probes, weakening the SERS signal at 2226&#xa0;cm^− 1. The aptasensor exhibited a favorable linear range from 0.001 to 100 mg L^− 1, with a detection limit of 0.39 × 10^− 3 mg L^− 1 toward tryptamine. The fabricated sensor was practically applied to the detection of tryptamine in liquor, white wine and vinegar samples, with results highly consistent with high performance liquid chromatography data, demonstrating the broad prospects of the developed analytical method in food threat detection.</p>