Nanofibers have long been exploited as a versatile, attractive material compared to their bulk counterparts due to their exceptionally high specific surface area and aspect ratio, highly porous structure, and wide range of materials that can be used for synthesis. Such unique properties helped exhibit their potential in a variety of applications in areas of energy, healthcare, and environment. Applications in energy are widely studied especially because of the global need for a source of energy and method of energy production that is alternative to the combustion of fossil fuels, as it presents many environmental drawbacks, which ultimately lead to adverse health effects on humans. To bypass the use of fossil fuels, renewable sources such as wind, hydro, geothermal, and solar energy have been introduced; devices that take advantage of alternative sources of energy such as nanogenerators, fuel cells, and thermoelectric generators have been developed.In this work, hybrid nanofibers were fabricated with precise control of the working parameters to fine tune their properties for various applications, including piezoelectrics and enzymatic fuel cell electrodes. Pure polyacrylonitrile (PAN) nanofibers were first electrospun based on a series of design of experiments to minimize both the diameter and beading. By correlating the solution, electrospinning, and environmental conditions to the resulting solution and nanofiber properties, it was found that not only physical properties such as electrical conductivity but also electrochemical properties of the nanofibers could be controlled. Physical and electrochemical properties of porous carbonaceous nanofibers derived from PAN, zinc acetate and multi-walled carbon nanotubes were studied with SEM, 4-point probe measurement, CV, LSV, and EIS. Based on the electroanalytical methods, the best nanofiber mats were chosen and used as electrodes to immobilize multienzyme cascade systems, a concept of which was demonstrated with a glucose oxidase-BglA bienzymatic system on a customized DNA scaffold as the anodic biocatalyst.