<p>Multifunctional nanocomposites have attracted extensive attention in the design of new SERS substrates. In this work, a metal-organic framework (MOF) modified with Ag nanoparticles (Ag NPs) and cysteine functionalized Fe₃O₄ (UiO-66-NH₂/Fe₃O₄/Ag) was prepared as a SERS substrate (UFAs). The substrate combines the enrichment ability of UiO-66-NH₂, the magnetic separation ability of Fe₃O₄, and the localized surface plasmon resonance (LSPR) effect of Ag NPs to achieve efficient detection of the target analyte. The results of adsorption kinetics showed that the adsorption process of malachite green (MG) was mainly dominated by chemical adsorption. In addition, we further explored the detection mechanism of UFAs for MG. UFAs enriched cation analytes such as MG through π-π stacking and electrostatic interaction and performed SERS detection. The UFA SERS substrate exhibits good detection sensitivity for MG, with a limit of detection (LOD) of 1.35 × 10⁻¹⁰ M (at 1619 cm⁻¹), and the SERS substrate has good uniformity and stability. The SERS substrate was applied to the detection of MG in aquaculture water. The recovery of MG in the sample was 91.2–105%, and the relative standard deviation (RSD) was 3.76–6.06%. This high-performance UFA SERS substrate also has great potential for the detection of food and other environmental pollutants in practical applications.</p> Graphical Abstract <p></p>