The aim of this study is to develop an electrospun calixarene nanofiber based calixarene for controlled release of hydrophobic drugs. To accomplish this, we have synthesized 5,17-bis-[Methyl-N-Methyltranylate]-25,26,27,28-tetrahydroxycalix[4]arene (Compound 3) of which nanofiber (F-14) was produced by electrospinning. The fabricated multilayered electrospun nanofiber was first characterized in terms of morphology and then drug loading and release kinetics were studied in different physiological pH. For this purpose, we have selected two fluorescent drugs which are thiabendazole (Tbz) and donepezil (Dnp) as model drugs to show the usage of the synthesized nanofiber in drug delivery system. Drug loading and release kinetics were monitored by using fluorescence spectroscopy. According to the results, maximum amount of loaded Dnp onto nanofiber was found to be as 30.529 μg in 20 mM Tris buffer, pH 7.4 end of 120 min. Data showed that loading amount of Tbz onto the nanofiber was measured to be as 1.688 μg to the 2.25 cm 2 of surface in 20 mM of Tris buffer, pH 7.4 at the end of 120 min. While max release of Dnp from nanofiber was also 9.720 μg at pH 2.2, that of Tbz from nanofiber was 0.243 μg at pH 7.4 at the end of 90 min. Drug loading to nanofibers was clarified by SEM, TEM, EDX and FT-IR analysis.