Theoretical modeling calculations and experimental measurements were adopted to investigate the oxygen activation effect of carbon nanofibers (CNFs) interacting with polypyrrole (PPY). The CNF undergoes a hydrothermal oxidation process to form epoxy and hydroxyl groups containing carbon nanofibers (CNF-O). The oxygen activation effect of CNF on the electronic and electrochemical properties was investigated through the interfacial interaction between CNF-O and PPY. Theoretical modeling calculation discloses that CNF-O/PPY exhibits lower electronic bandgaps (0.64 eV), a higher density of states (10.039 states/eV), and a lower HOMO–LUMO molecular orbital energy gap (0.077 eV) than CNF/PPY (1.56 eV, 7.946 states/eV and 0.112 eV), presenting its superior electronic conductivity and electroactivity. The Mulliken population and charge density difference analysis disclose the stronger interface interaction of CNF-O/PPY caused by epoxy and hydroxyl groups. Cyclic voltammogram measurements reveal that CNF-O/PPY exhibits a higher response current and a higher specific capacitance (221.1–112.2 mF g−1) than CNF/PPY (57.6–24.2 mF g−1) at scan rates of 5–200 mV s−1. Electrochemical impendence spectrum measurements disclose that CNF-O/PPY exhibits a lower charge transfer resistance (0.097 Ω), a lower ohmic resistance (0.336 Ω), a lower Warburg impedance (317 Ω), and a higher double-layer capacitance (0.113 mF) than CNF/PPY (1.419 Ω, 9.668 Ω, 7865 Ω, and 0.015 mF). Both theoretical and experimental investigations prove that CNF-O/PPY presents an intensified intermolecular interaction rather than CNF/PPY. The promotive oxygen activation effect of CNF could contribute to improving the electronic and electrochemical properties of CNF-O/PPY.