Biomass is an interesting renewable energy resource as it is widespread in nature and low cost. The development of bio-oil derived from biomass as a fuel is still a scientific and industrial challenge. In this context, we demonstrate the synthetic method of bio-oil upgrading catalysts based on hierarchical zeolites and open up interesting perspectives for bio-oil upgrading processes. The hierarchical ZSM-5 zeolite has been successfully prepared via a direct hydrothermal synthesis with the aid of a commercial organosilane surfactant (TPOAC). The influences of TPOAC content and Si/Al ratio on hierarchical structures were also systematically studied. To illustrate their catalytic performances, an esterification reaction of various organic acids such as (acetic acid and levulinic acid) and alcohols was performed as the model reaction representing the bio-oil upgrading application. The synergic effect of acidity and the hierarchical structure of catalysts can greatly enhance the catalytic performance in terms of activity, product yield, coke formation, and reusability of the catalysts. For example, they can convert almost 100% of reactant in 8 h in the esterification of acetic acid and alcohols, whereas the conventional zeolite reveals significantly lower activity (<20%). Interestingly, the hierarchical zeolite can also greatly improve the catalytic activity of the esterification of levulinic acid and ethanol to produce ethyl levulinate that can be used as a diesel miscible biofuel (DMB). In addition, the efficiency of hierarchical catalysts obtained by different synthesis methods is also discussed. This first example demonstrates that the hierarchical zeolite obtained via a direct synthesis approach can benefit bio-oil upgrading applications via the esterification of various carboxylic acids.