In the present study, the tri-layer nanofibers were synthesized via triaxial electrospinning process to control the sustained delivery of Doxorubicin (DOX), Paclitaxel (PTX) and 5- fluorouracil (5-FU) anticancer drugs from nanofibers. The 5-FU molecules were incorporated into the core solution (chitosan/polyvinyl alcohol (CS/PVA)) to fabricate the CS/PVA/5-FU inner layer of nanofibers. The intermediate layer was prepared from poly(lactic acid)/chitosan (PLA/CS) nanofibers. The DOX and PTX molecules were initially loaded into the g-C3N4 nanosheets and following were incorporated into the PLA/CS solution to fabricate the outer layer of nanofibers. The synthesized nanosheets and nanofibers were characterized using XRD, SEM, TEM and UV-vis analysis. The PLA/PVA/CS/FU/g-C3N4/DOX/PTX single layer nanofibers were also synthesized via electrospinning method. The drug loading efficiency, degradation rate and anticancer drugs release profiles from single layer and tri-layer nanofibers were investigated under various intermediate and shell layer thicknesses. The pharmacokinetic studies were performed to understand the drugs release mechanism from nanofibers. The cell viability and cell attachment of drug loaded single layer and tri-layer nanofibers toward the MCF-7 breast cancer cells were examined to achieve an optimum nanofibrous formulation for the breast cancer treatment. The obtained results revealed the high activity of tri-layer nanofibers for the breast cancer cells killing.