A certified reference material (CRM) was characterized for the determination of copper (Cu) in bio-ethanol samples. The material for the CRM was made from rice by fermentation and refined by distillation, which was further added with Cu acetate and acetic acid. Because the ionization of Cu in inductively coupled plasma mass spectrometry (ICP-MS) was affected by the content of ethanol in the sample, matrix matched methods were developed for the determination of Cu, where the bio-ethanol samples were 10-times (v/v) diluted to 1 mol L-1 nitric acid solution. All of the standard solutions for calibration were also adjusted to 1 mol L-1 nitric acid solution with 10% (volume fraction) ethanol. Determination of the concentration of Cu in the sample was carried out by gravimetric standard addition ICP-MS, isotope dilution (ID-) ICP-MS, and graphite furnace atomic absorption spectrometry (GF-AAS). The property value of Cu was calculated based on the weighted mean of the observed values by these methods, with the reciprocal of the uncertainty of each method as the weight. The deviation factor of sampling for characterization was also taken into consideration for calculating the certified value. The combined uncertainty (3.79%, relative value) of the certified value was calculated as a combination of the uncertainties of homogeneity (3.53%), analytical method (0.64%), method-to-method variance (1.16%), and calibration standard (0.35%). The certified value of Cu in the CRM was (53.7 +/- 4.1) ng g(-1) (property value +/- expanded uncertainty, k = 2), which is traceable to the International System of Units (SI units) via the Japan Calibration Service System (JCSS). (C) 2012 Elsevier Ltd. All rights reserved.