In this study, a systematical technique has been developed to experimentally and numerically evaluate enzyme-assisted hot waterflooding performance in a heavy oil reservoir for the first time. Experimentally, an enzyme solution (i.e., a protein-based liquid catalyst) is prepared and used to displace heavy oil in sandpacked experiments at elevated temperatures, during which pressures and fluid productions are continuously monitored and measured. Numerically, reservoir simulation is performed to reproduce the experimental measurements and then extended to evaluate the performance in a targeted heavy oil reservoir. Once history matching on the experimental measurements is completed, such a calibrated model is then employed to optimize enzyme concentration, temperature, and aging time, respectively. It is found from the displacement experiments that temperature imposes a significant impact on heavy oil recovery with its appropriate range of 45–55 °C, and enzyme positively contributes to heavy oil recovery for most scenarios. Compared to the traditional waterflooding mechanisms, the enzyme-assisted hot waterflooding process shows its considerable potential in heavy oil recovery by means of reducing oil viscosity, altering wettability, and reducing interfacial tension.