Bio-oil obtained through the gasification or pyrolysis of biomass is a renewable energy source with the potential to be used in motor vehicles. However, when the properties of bio-oil are compared to crude oil, bio-oil is observed to have high oxygen content and acidity. The aim of this study is to enhance the physical properties of bio-oil and produce new alternative fuels to crude oil. For this purpose, nickel and cobalt-incorporated mesoporous HZSM-5 catalysts have been synthesized. The synthesized catalysts were characterized by X-ray diffraction, N2 adsorption–desorption, Scanning electron microscopy energy dispersive spectroscopy, Inductively coupled plasma optical emission spectroscopy, Fourier-transformed infrared spectroscopy, and thermogravimetric/differential thermal analysis. In the study, formic acid, furfural, and hydroxypropanone were used as model components. To enhance catalyst activity, nickel was loaded onto the HZSM-5 catalyst. However, during biofuel production, a significant amount of coke was formed as a by-product. Therefore, cobalt was impregnated to reduce coke formation. In the activity test studies, a conversion in the range of 77–84% was achieved with HZSM-5 catalysts. Nickel addition increased the paraffin and olefin content in the biofuel along with bio-oil conversion. The maximum paraffin selectivity (97%) was provided with the 5Ni@HZSM-5 catalyst. However, the highest biofuel selectivity (77.5%) with the minimum coke formation (4%) was observed with the 5Co-5Ni@HZSM-5. In the study, the regeneration and long-term catalytic activity were also investigated, and the results showed that 5Co-5Ni@HZSM is an attractive catalyst for biofuel production from bio-oil.