Recently, due to the impressive chemical properties of nanofibers, many efforts have been made to develop their applications. In this study, polycaprolactone (PCL), phenytoin, and collagen nanofibers extracted from Catostylus mosaicus jellyfish were fabricated using the electrospinning technique. PCL solution with a concentration of 10% (w/v) and collagen solution with a concentration of 1% (w/v) were prepared at room temperature. The electrospinning solution was prepared with a volume ratio of 70:30 PCL solution to collagen solution. Phenytoin with a concentration of 1 wt% was added to the PCL-collagen solution. Electrospinning was performed under 15 kV voltage, 0.7 mL/h flow rate, and 15 cm needle-collector distance. The morphology, molecular structure, and thermal stability of the nanofibers were examined using field emission scanning electron microscopy (FE-SEM), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA) experiments, respectively. Moreover, swelling and drug release experiments were performed. Based on the results, uniform and asymmetric PCL-collagen-phenytoin nanofibers with an average diameter of 225 ± 21 nm and a swelling percentage of 293% were produced. FTIR analysis showed the presence of hydrogen bonding between PCL and collagen. The TGA results confirmed the physical interaction between collagen and PCL. The release of phenytoin initially shows a burst release followed by a sustained release. The results revealed that the produced nanofibers can be a suitable candidate for use in biomedical applications.