Due to the environmental problems caused by fossil fuels, it is necessary to choose an alternative source of energy for these fuels. One of the best sources for this is biofuels. This study produced biofuels from Sargassum macroalgae from the Persian Gulf using a hydrothermal liquefaction process. By examining the operating parameters of temperature, residence time, and biomass to solvent ratio, the optimal amount of each of these parameters was selected to achieve maximum biofuel yield. This optimization was performed by designing an experiment with the response surface method, by Design-Expert software. To provide an experimental equation and predict biofuel yield under different operating conditions, software were proposed the inverse model and quadratic equation. In this model, the regression of equations was 0.9837 and, the p-value was 0.0006. The optimization of this process, the maximum yield of biofuel was predicted to be 25% wt., which is obtained at a temperature of 350 ° C, a residence time of 35 minutes and, biomass to solvent ratio of 8.6% wt. By repeating these conditions in the laboratory, the maximum biofuel yield was 26% wt. This yield confirms the conditions predicted by the software. At the optimum process, all of the products were characterized. The Heating value of the biofuel produced was 33.91 MJ/Kg. This amount is due to the high content of carbon in biofuel. The distillation temperature of 34.5% of the constituents of biofuels is similar to the heavy naphtha, which indicates the presence of large amounts of light compounds in biofuels. The maximum evaporation of biofuel organic compounds occurred at a temperature of 235 ° C and indicates the high quality of biofuels. It was also found that most of the compounds of biofuels were cyclic different groups of Cyclopenten, o-Xylene, Heptyne, Cyclohexanone, and Diazocine. The high amounts of carbon and low nitrogen in the solid phase led to a heating value of 5.61 MJ / Kg, which makes this product