This study evaluates a simultaneous assessment of organic matter (OM) and trace elements (TE) bio-accessibility in substrate and digestate from a full-scale anaerobic digester by a sequential OM extraction method. Simultaneous release of TE was determined along with the extraction of different OM fractions and the effects of extracting reagents on characteristics of OM were evaluated by nuclear magnetic resonance (NMR) spectroscopy. The reagents used for sequential extraction of OM were not enough selective. However, proteins were particularly removed by 0.1 M NaOH, while 72% H2SO4 mainly extracted hemicellulose and cellulose. The OM fractionation allowed for simultaneous extraction of greater than60% of total As, Cd, Co, Fe, Mn, Ni and Zn, while the extraction was limited for Al, Cr, Cu, Mo, and Pb. In substrate, greater than50% of total As, Co, Mn and Ni and less than40% of total Fe, Zn and Mo were identified in bio-accessible fractions. In digestate, all elements demonstrated poor bio-accessibility except for As.less thanbr /greater than (Copyright © 2019 Elsevier Ltd. All rights reserved.)