Carbon nanofiber/manganese oxide (CNF‐MnO) hybrid nanofibers are synthesized by calcination of potassium permanganate (KMnO4) loaded bacterial cellulose (BC) hydrogels. The chemical structure, morphology, performance, and application of CNF‐MnO aerogels are characterized and studied. The results revealed that MnO nanoparticles are uniformly deposited on the surface of CNF which derived from BC hydrogels. An amperometric superoxide anions (O2•−) sensor is fabricated by the immobilization of the CNF‐MnO aerogels on a glassy carbon electrode, which displays a linear amperometric response with a high sensitivity of 76.2 µA cm−2 × 10−3m−1 and a low detection limit of 1.2 × 10−9m in the concentration range of 5.0 × 10−9m – 2.5 × 10−6m. The successful detection of O2•− released from cancer cells verifies the potential application in biomedical field.