According to the directives for the work of reference materials GB/T 15000,nicotine certified reference material was prepared.Nicotine was prepared by extraction,column chromatography and steam distillation.Its chemical structure was identified by UV，IR，MS and NMR method.Its homogeneity and stability were systematically checked.A cooperative certification was conducted by 8 laboratories，and the results of certification for nicotine were analyzed.The homogeneity and stability of the nicotine reference material were good,and the certified value was 99.12% with expanded uncertainty of 0.64% in confidence coefficient of 95%.The reference material of nicotine conforms to directives for the work of reference materials,and it could be used for method validation and quality control of regarding products.