The nanofibers fabricated by using an electrohydrodynamic process has been used as various applications, such as nano-device, filtering system, protective clothes, wound dressing, and drug delivery system (DDS). Of these applications, the DDS should be needed to minimize side effects of drugs, maximize the properties of medicine, and efficiently deliver the required amount of drugs to the diseased area. In this paper, by using the electro spinning process, which is one of electrohydrodynamic processes, two different types, polycarprolactone and poly(ethylene oxide)/Rhodamine B, of electrospun mats were fabricated layer by layer and the release behavior of Rhodamine B was characterized with time. In addition, to show the feasibility of DDS of this type, we tested release behavior of a peptide of the nanofiber system, a PCL/(Peptide+PEO)/PCL nanofiber mat. The released peptide did not loss biological activities. From these results, we believe that the layered nanofiber mat as a DDS has enough function of a new drug delivery system.