Cellulose monoacetate/Nafion (CMA/N) hybrid nanofibers were produced via a one-step electrospinning method. Nanofibers morphologies transformed from uniform to bead on a string defect morphology with increasing Nafion ratio in CMA/N hybrid nanofibers. The melting point of CMA was detectable at DSC measurement, but since the addition of Nafion did not allow a proper crystallization of CMA, melting peak disappeared after the Nafion addition. Decomposition temperature decreased dramatically with the addition of Nafion into CMA/N nanofibers and decomposition took place at a broad temperature range. Nanofibers were also electrospun on the cylindrical graphite electrode for DNA electrochemical sensor analysis. Unmodified and NH-modified single strand DNA molecules were immobilized via physical adsorption method on the asprepared nanofiber sensory system. Electrochemical analysis were performed via differential pulse voltammetry (DPV) to observe the guanine oxidation signal at unmodified and NH-modified DNA. Maximum oxidation signals were detected from pure CMA nanofibers at unmodified DNA. Signal intensity increased with the addition of Nafion into CMA/N nanofibers at NH-modified DNA sample comparing to unmodified DNA. It was concluded that DNA molecules could be properly immobilized on the produced CMA/N hybrid nanofibers via physical adsorption method and used as electrochemical DNA biosensor.