Synthesis of NiSn alloy nanoparticle-incorporated carbon nanofibers was performed by calcining electrospun mats composed of nickel acetate, tin chloride and poly(vinyl alcohol) under vacuum. The electrochemical measurements indicated that utilization of tin as a co-catalyst could strongly enhance the electrocatalytic activity if its content and calcination temperature were optimized. Typically, the nanofibers prepared from calcination of an electrospun solution containing 15 wt% SnCl2 at 700 °C have a current density almost 9-fold higher than that of pristine nickel-incorporated carbon nanofibers (77 and 9 mA/cm2, respectively) at 30 °C in a 1.0 M urea solution. Furthermore, the current density increases to 175 mA/cm2 at 55 °C for the urea oxidation reaction. Interestingly, the nanofibers prepared from a solution with 10 wt% of co-catalyst precursor show an onset potential of 175 mV (vs. Ag/AgCl) at 55 °C, making this proposed composite an adequate anode material for direct urea fuel cells. Optimization of the co-catalyst content to maximize the generated current density resulted in a Gaussian function peak at 15 wt%. However, studying the influence of the calcination temperature indicated that 850 °C was the optimum temperature because synthesizing the proposed nanofibers at 1000 °C led to a decrease in the graphite content, which dramatically decreased the catalyst activity. Overall, the study opens a new venue for the researchers to exploit tin as effective co-catalyst to enhance the electrocatalytic performance of the nickel-based nanostructures. Moreover, the proposed co-catalyst can be utilized with other functional electrocatalysts to improve their activity toward oxidation of different fuels.