Abstract Catalytic hydrotreatment of pyrolysis-oil from biomass is an important process to improve oil characteristics for use as liquid fuel. Bimetallic NiCu catalysts are currently attractive for use in hydrotreatment process due to its highly active for hydrogenation, hydrodeoxygenation and hydrocracking reactions. In this study, NiCu catalyst supported by several mono-oxide (i.e. γ-Al2O3, ZrO2, SBA-15 and MCM-41) and mixed-oxide (ZrO2-SBA-15 and ZrO2-MCM-41) materials was tested for hydrotreatment of guaiacol (as pyrolysis-oil model compound) and pyrolysis-oil from eucalyptus under several reaction temperatures (200–350 °C) and times (1–6 h). Among all catalysts, NiCu/ZrO2-SBA-15 showed the highest activity toward the hydrotreatment of guaiacol, from which hydrodeoxygenation yield of 87.3% with relatively low carbon deposition (3.4 wt%) was achieved from the reaction at 325 °C for 2 h. For the hydrotreatment of pyrolysis-oil in the presence of NiCu/ZrO2-SBA-15, ungraded oil with favorable qualities (i.e. high H/C ratio and low thermogravimetric residue) was obtained from the reaction at 350 °C for 3 h. After reaction test, catalyst regeneration and reusability were also studied. The regeneration of spent NiCu/ZrO2-SBA-15 by oxidation with O2 at 600 °C for 3 h can remove most of carbon species from catalyst surface with insignificant change in catalyst surface properties. In addition, the regenerated catalyst can be reused for at least 5 reaction cycles without significant deactivation observed.