Water emulsified heavy fuel oil (HFO) has been a promising alternative fuel for reducing oil consumption and preventing environmental pollution. However, the intrinsic challenges such as fuel formula, emulsion stability, and preparation process normally limit its further applications in energy-saving and emission reduction applications. In this study, the glucose obtained from biomass was added to a dispersed-phase aqueous solution of water emulsified HFO to prepare a novel alternative emulsified fuel. First, based on the preliminary experimental design, the effects of glucose and surfactant on the stability of the HFO emulsion were systematically evaluated through the appearance of emulsion separation, droplet size distribution, and rheological characteristics. It indicated that the surfactant ratio, hydrophilic-lipophilic balance value, solution ratio, and glucose/water ratio had significant impacts on emulsion stability. Subsequently, the optimum range of influencing factors of emulsion stability was determined by a single factor experiment and determined by the response surface methodology based on the Box-Behnken design; the optimal values of the above factors were 2.439 v/v%, 5.807, 26.462 v/v%, and 35.729%, respectively. Under these conditions, an optimal glucose solution emulsified HFO with a uniform brown color and long-term stability was obtained, making the unseparated emulsion ratio reach 98% (lasting for 7 days at 85 °C). Meanwhile, it emerged that the influence of multifactor on emulsion stability was not a simple linear correlation, and there were significant interactions between the solution ratio and the surfactant ratio, as well as between the glucose/water ratio and the surfactant ratio.