Tuning the reactivity of graphene enables molecular-level engineering of the lattice, achieving desired chemical and structural properties through functionalization, doping, and etching. Atom-thin graphene film hosting Å-scale pores, with capability to differentiate molecules with sub-Å resolution, is ideal to advance performance for challenging molecular separation. Control over pore formation is needed to improve pore size distribution (PSD), in particular, to increase the percentage of molecular selective pores. An attractive approach is to modulate the energy barriers involved in the pore formation to control PSD. In this study, it is shown that electron-hole puddles induced in graphene by the underlying Cu substrate increase its reactivity toward O3. These puddles promote electron transfer during O3 chemisorption and reduce the energy barrier for lattice gasification. This strategy is implemented to increase the density of molecular-selective pores by expanding small non-permeable pores. The resulting porous graphene membranes demonstrate highly promising separation performance for the CO2/N2 gas pair. This approach provides a new pathway to finely control pore formation for advanced applications in molecular separation and beyond.