Malondialdehyde (MDA) is a biomarker of lipid peroxidation and intracellular oxidative stress and can also be used to monitor food freshness. However, fast, accurate and ultrasensitive detection of MDA remains a challenge. In this work, a chemical reaction tandem surface-enhanced Raman spectroscopy (SERS) method was applied for trace MDA detection by gold nanoparticles (AuNPs) enriched on the surface of Q sepharose spheres ([email protected]). After derivatization of MDA with 2-thiobarbituric acid (TBA), TBA-MDA adduct could be directly detected by SERS in the reaction media utilizing [email protected] as SERS substrate. QSS play multiple roles as AuNPs adsorbent, AuNPs carrier against aggregation in acid environment, and TBA-MDA adduct enricher. SERS detection in solution and the micro-scale homogeneous AuNPs distribution on QSS surface contribute to the improved reproducibility of the substrate. This method has a wide linear range (0.33 μM to 3.3 mM in a liquid environment), low limit of detection (∼10−10 M at dry state) and has already been applied in the direct determination of MDA in oil samples without standard addition.