Aflatoxin B 1 in a peanut oil matrix certified reference material (CRM, GBW10176) has been issued for accurate determination of aflatoxin B 1 in real samples. This matrix CRM was prepared from commercial peanut oils naturally contaminated by aflatoxin B 1 , with the aid of a liquid−liquid extraction process. Specially, the certified value of CRM was determined using two individual approaches, liquid chromatography−isotope dilution tandem mass spectrometry and standard addition liquid chromatography−fluorescence detection techniques. More importantly, assessments of homogeneity and short-and long-term stabilities confirmed that the GBW10176 CRM was sufficiently homogeneous between packages or within package, and it could be stable up to 7 days stored below 50 °C or 18 months at 4 °C. The metrological traceability of GBW10176 was ensured by the application of pure aflatoxin B 1 (GBW10172). Its uncertainty was evaluated with the contributions from the characterization, homogeneity, and stability. Resultantly, the certified value of the aflatoxin B 1 CRM (GBW10176) was 23.4 μg/kg with an uncertainty of 3.1 μg/kg (k = 2).