Abstract: To fulfill the requirements of food safety monitoring in China, a certified reference material (CRM) for aflatoxin B₁ (AFB₁) analysis in peanut oil was developed. This CRM was derived from naturally contaminated positive samples, undergoing processes such as blending, nitrogen purging, and packaging. Homogeneity of the CRM candidate was assessed using High-Performance Liquid Chromatography with Fluorescence Detection (HPLC-FLD), while its stability was evaluated using High-Performance Liquid Chromatography - Isotope Dilution Mass Spectrometry (HPLC-ID-MS/MS). Value assignment was performed through collaborative laboratory efforts utilizing both HPLC-FLD and HPLC-ID-MS/MS. The results indicated that the CRM meets technical specifications, displaying excellent homogeneity and stability for 16 months at −18 ℃. The certified value of AFB₁ is 15.6 μg/kg with an expanded uncertainty of 1.6 μg/kg (k = 2). The uncertainty assessment encompassed factors from homogeneity, stability, and value assignment. The developed CRM for AFB₁ in peanut oil, certified as GBW(E) 100604, fulfills the urgent need for traceable standards in edible oil AFB₁ measurement and ensures accurate and reliable results.