By comparing the two types of activated carbon fibers (ACFs), characteristics of adsorption sites for nitrate ion other than quaternary nitrogen (N-Q) were investigated. Using phenol resin as precursors, activation with ZnCl₂ was performed, and then heat treatment at 950 °C was carried out to prepare ACFs without N-Q, while ACFs with N-Q was prepared in the same method using polyacrylonitrile-based carbon fiber as precursors. We assessed the amount of functional groups, elemental composition, porous properties, and model of unit crystal size of graphene. For both ACFs with N-Q and without N-Q, equilibrium adsorption amount was not always simply proportional to surface area, but to the average number of benzene rings (Bz-rings) of graphene universally. PhR-5.0Z4 had only 20 benzene rings per graphene unit, but after heat treatment at 950 °C, the number drastically increased to 1088 (PhR-5.0Z4-9.5HT30). However, when the ACFs contained a large amount of oxygen, the number of Bz-rings was limited to 792 (PhR-5.0Z4-Ox-9.5HT30) even after heat treatment at 950 °C, and did not increase sufficiently. Cπ sites are more susceptible to oxygen inhibition than N-Q in adsorbing nitrate ions. For ACFs having Cπ sites as main adsorption sites, the heat treatment at 950 °C without oxidation can enhance the nitrate ion adsorption capacity.