The mechanism of nucleophilic substitution deserves more investigation to include more reaction systems such as two-dimensional (2D) materials. In this study, we used fluorinated graphene (FG) as a representative 2D material to reveal the in-depth mechanism of its defluorination and nucleophilic substitution reaction under attack of common nucleophiles to explore the chemistry of 2D materials and enrich the research on the nucleophilic substitution reaction. DFT calculations and electron paramagnetic resonance spectroscopy (EPR) demonstrated that defluorination of FG occurred via a radical mechanism after a single electron transfer (SET) reaction between the nucleophile and C–F bond, and a spin center was generated on the nanosheet and fluorine anion. Moreover, neither the S_N1 nor S_N2 mechanism was suggested to be appropriate for the substitution reaction of FG with a 2D structure due to the corresponding kinetics or thermodynamics disadvantage; hence, its nucleophilic substitution was proved to occur via a radical mechanism initiated by the defluorination step. The proposed substitution mechanism of FG demonstrates that nucleophilic substitution via a radical mechanism can also be applied to the attacking process of common nucleophiles without any particular conditions. Furthermore, it has been discovered that triethylamine without active hydrogen can be covalently attached to graphene nanosheets via a nucleophilic substitution reaction with FG; this further indicates a radical process for the nucleophilic substitution of FG rather than an S_N1 or S_N2 mechanism. The detailed process of the nucleophilic substitution reaction of FG was revealed to occur via a radical mechanism depending on the 2D structure of FG, which could also represent the typical characteristic of 2D chemistry.