In this paper, we have fabricated poly( N-isopropylacrylamide)-co-polystyrene ( PNIPAM-co-PS) nanofibers by electrospinning and explored the possibility to utilize the PNIPAM-co-PS nanofibers to enhance the permeation and uptake of the anticancer drug daunorubicin in drug-sensitive and drug-resistant leukemia K562 cells. Our MTT assay and electrochemical studies demonstrate that PNIPAM-co-PS nanofibers could play an important role in facilitating the cell track and drug delivery to the cancer cells. Meanwhile, the observations of atomic force microscopy ( AFM) and confocal fluorescence microscopy indicate that the relevant interaction of the PNIPAM-co-PS nanofibers with bioactive molecules on the membrane of leukemia cell lines could affect the intracellular drug uptake positively and lead to the efficient accumulation of daunorubicin in drug-sensitive and drug-resistant cancer cells.