A simple method was developed for the preparation of ordered mesoporous silica-carbon composite nanofibers (OMSCFs). The OMSCFs exhibited high carbon content, continuously long fibrous properties, uniform accessible mesopores, and a large surface area. The OMSCFs were also found to have ion-exchange capacity. On the basis of the size-exclusion effect of the mesopores and mixed-mode hydrophobic/ion-exchange interactions, the OMSCFs were applied for rapid enrichment of endogenous peptides by using a miniaturized solid-phase extraction format. The adsorption mechanism was studied, and the eluting solution was optimized with standard peptide/protein solutions and protein digests. Employing a successive three-step elution strategy, followed by LC-MS/MS analysis, led to excellent performance with this approach in the extraction and prefractionation of peptides from human serum.