New carrier platforms have been designed for an electrospun pyridinium calixarene nanofiber for controlled drug delivery. First, 5,11,17,23-tetra-tert-butyl-25,27-bis(3-aminomethyl-pyridineamido)-26,28-dihydroxycalix[4]arene (3-AMP) scaffold was produced by electrospinning. AMP scaffold was modified by human serum albumin (HSA), folic acid (FA), and glutathione (GSH). Doxorubicin (DOX) was loaded to surfaces of the AMP, AMP-HSA, AMP-HSA-FA, and AMP-HSA-GSH nanofibers by using DOX solution in different buffers with, 2.2, 4.0, 6.0, and 7.4 pH. The release studies DOX from four different nanofibers was also done in a various amount microenviroments by changing pH values. The loading and release amount of DOX was estimated from the calibration curve drawn at 480 and 560 nm of excitation and emission wavelengths by using a fluorescence spectrophotometer. The loading studies were confirmed by Fourier transforms infrared, atomic force microscopy, transmission electron microscopy, scanning electron microscope, and energy-dispersive X-ray (EDX) analysis.