The synthesis of functional nanomaterials with unique structures and morphologies as efficient biocatalysts for sensing application has attracted tremendous interest. Herein, Fe3C nanoparticles encapsulated within nitrogen doped carbon (Fe3C/N-C) nanofibers have been prepared through a facile electrospinning strategy and a carbonization process. The resulting Fe3C/N-C hybrid nanofibers display a superior oxidase-like performance toward the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and other substrates, which is dependent on the formation of Fe3C nanoparticles and their crystallinity. The obtained Fe3C/N-C hybrid nanofibers-based oxidase-like catalyst shows a good long-term stability and reusability. Thanks to the unique catalytic activity for oxidase mimicking, an efficient sensing platform to sensitively determine sulfite and L-cysteine with low detection limits of 24.9 and 23.0 nM (S/N = 3), respectively, as well as excellent selectivity and anti-interference ability has been developed. This work demonstrates a versatile approach to fabricate Fe3C/N-C hybrid nanofibers as enzyme mimics with perfect catalytic efficiency, affording a facile and sensitive colorimetric approach for potential applications in biosensing and other biotechnologies.