The certified reference materials (CRMs) are necessary for accurate quantification and insurance of comparability and traceability of results. Patulin is a typical mycotoxin in a variety of food commodities. Here, patulin CRM GBW(E)100673 was characterised and its purity was assessed by two independent orthogonal approaches including mass balance (MB) and quantitative nuclear magnetic resonance spectroscopy (qNMR) methods. From MB equation, the calculated purity was 996.9 mg/g with subtraction of water, volatile solvent, inorganic and structurally related impurities. In the other qNMR method, the calculated purity was 996.7 mg/g. This CRM was homogeneous and stable for at least 9 months under -20 °C in dark. Finally, a purity of 997 mg/g with an expanded uncertainty of 3 mg/g ( k =2) was finally assigned to patulin CRM in this study. High-purity patulin CRM was fully characterised and assessed for the first time. The new CRM can be applicable to routine monitoring and risk assessment for assurance of accuracy results in food safety.