Hydrogen is considered as the most promising future fuel, as its combustion generates only water vapor besides energy. However, finding efficient, safe and low cost storage methods is the basic impediment for the adoption of hydrogen as fuel. For this purpose, five biomass-based carbon samples have been successfully prepared through KOH activation procedure. The microstructure of the prepared materials was tuned by varying KOH/precursor weight ratio from 1:1 to 5:1, in order to optimize their hydrogen storage behavior and to clarify the storage mechanism. A careful textural and morphological characterization of the prepared samples (N2 and CO2 adsorption, X-ray diffraction, electron microscopy observations) showed that by increasing the activation ratio the nature of the carbons changes from microporous to micro-mesoporous. Thus, an increase in the surface area was observed which enhanced H2 sorption capacity when high H2 pressures were adopted. At subatmospheric pressure, the role of active sites for H2 adsorption, located in the oriented graphene sheets, was clearly elaborated. Finally, the optimal carbon sample has shown a capacity of 6wt% and 1.22wt% at −196°C and 25°C respectively, and 200bar. These results make biomass-based carbons promising materials for H2 storage application.