To reduce the environmental footprints of fossil fuels, there is a need to source more biomasses to increase the renewable energy supply. However, a critical study of the energy-conversion process of biomass must be carried out to make the process economical. In this research, the optimization of the bio-oil production from the thermal conversion of novel biomass- cow hoof was carried out. Three independent variables- temperature of pyrolysis, heating rate and CaO catalyst mass were studied at 3 levels based on the rotatable central composite design (CCD) of the response surface methodology (RSM) to ascertain their influence on two responses- bio-oil yield and its HHV. The quadratic model was more suitable to fit the experimental data. At optimum values of the process variables, bio-oil yield of 50.64% and HHV of 23.86 MJ/kg were obtained. From the analysis of variance carried out, the model R2 values were 0.9949 and 0.9802 respectively for the bio-oil yield and HHV models which showed the models’ ability to predict the bio-oil yield and its HHV in the pyrolysis process is high. The characterization of the bio-oil revealed it has better fuel properties compared with most bio-oils from some biomasses hence it is a viable renewable energy source.