Bio-oil can be used as a substitute for fossil fuels after it is upgraded. Bimetal-modified HZSM-5 catalysts with various Ni-to-Co ratios were prepared to address catalysis problems, including deactivation of the catalysts and low hydrocarbon yields. The catalytic performance of Ni–Co/HZSM-5 in upgrading the simulated bio-oil was investigated with a fixed-bed reactor, and the influence of the loaded duplex metal ratio was also discussed. The new moderately strong/strong acid sites of Ni–Co/HZSM-5 changed according to the Ni/Co loading ratios, which substantially affected the acidity, catalytic activity and selectivity of the Ni–Co/HZSM-5. However, incorporating Co and Ni into the zeolite did not alter the structure of the HZSM-5. The interactions of the loaded bimetallic oxides reached equilibrium in 6Ni–4Co/HZSM-5, in which moderately strong acid sites and strong acid sites were formed after loading with Co₃O₄ and NiO. With a suitable acid site ratio, 6Ni–4Co/HZSM-5 exhibited excellent performance, with a lower coke deposition of 3.29 wt% and stable catalytic activity, and the conversion remained at 83–73% during 360 min of uninterrupted catalysis. Periodic changes in the acid sites and interfacial protons were the critical factors that improved the properties of the 6Ni–4Co/HZSM-5 and enhanced its selectivity for aromatic compounds.