Bio-oil, produced by the pyrolysis of cheap and renewable biomass, could be upgraded into transportation fuels, therebying achieve carbon emission reduction. Herein, we show a coagulation bath technique to prepared millimeter-scale N-doped carbon sphere with in-situ coated γ-Al2O3 supported Co NPs catalysts (Co/NCAn), with efficient hydrodeoxygenation activity and mechanical strength. Using guaiacol as a model substrate, Co/NCA2.5 outperformed other catalysts in terms of guaiacol conversion (100%), cyclohexane selectivity (93.1%) and deoxygenation rate (93.3%). The abundant N species in the micro-mesoporous carbon sphere promoted the increase of cyclohexanol, and the appropriate amount of Lewis acid accelerated the cleavage of C–O linkages, thus demonstrating a significant selectivity to alkanes, especially to cyclohexane. The Co atom anchoring sites on the N-doped carbon sphere catalyst was unraveled by the DFT simulation. Using a straw-derived bio-oil feedstock, this catalyst performed upgrading of oxygenated substrates into liquid hydrocarbon fuel with high efficiency. Consequently, the Co-supported carbon sphere catalyst having proper acid sites and stable micro-medium pore distributions is advantageous for implementing economically bio-oil deoxidation upgrading.