Recently, metal-organic frameworks (MOFs) based substrates have shown great potential for the quantitative analysis of food samples by surface-enhanced Raman scattering (SERS) due to their unique properties. Herein, we developed two UiO-66 MOFs/gold nanoparticles (AuNPs) based substrates by self-assembly, including UiO-66/AuNPs suspension substrate and UiO-66(NH2)/AuNPs/Nylon-66 flexible membrane substrate, for quantitative analysis of complex food samples by SERS. UiO-66/AuNPs suspension substrate was prepared for SERS-based determination of a carcinogenic heterocyclic amine in barbecue meat. UiO-66(NH2)/AuNPs/Nylon-66 membrane substrate was fabricated for the simultaneous separation, enrichment, and in situ analysis of Sudan Red 7B in chilli products. The heterocyclic amine and Sudan dye in real samples could be detected and quantified with the recoveries of 82.3–110% and 84.5–114% and relative standard deviations (RSDs) of 3.1–11.0% and 1.9–5.6% (n = 3) by use of these two substrates, respectively. These two UiO-66/AuNPs based substrates combined molecular enrichment and SERS activity, achieving excellent analytical accuracy and widening SERS application in practical food safety analysis.