The invention relates to a preparation method of esterified biological asphalt. The preparation method comprises the following steps: 1) mixing bio-oil with alcohol, and adding a catalyst to perform esterification reaction; 2) extracting the esterified bio-oil obtained in the step 1) with water to obtain an insoluble matter; and 3) performing oxidation treatment on the insoluble matter obtained in the step 2) to obtain the esterified biological asphalt. By adopting the preparation method provided by the invention, heavy components of biomass oil can be effectively utilized and turned from wastes into treasure; and furthermore, by improving the process, the fuller reaction of alcohol and bio-oil can be promoted, and the esterification efficiency of the biological asphalt is improved. The prepared biological asphalt has the characteristics of low viscosity increasing speed, good aging resistance and the like.