Background: Chemotherapy as an important tool for cancer treatment faces many obstacles such as multidrug resistance and adverse toxic effects on healthy tissues. Drug delivery systems has opened a new window to overcome these problems. There has been a strong interest development of new platform and system for delivof chemotherapeutic agents. Purpose: In the present study, a green synthesis method was chosen and performed for preparation of a novel amphoteric calix[4]arene (Calix) macrocycle with low toxicity to the human body. Materials and methods: The amphoteric Calix was coated on the surface of Fe₃O₄ magnetic nanoparticles and used as a magnetic nanocarrier for simultaneous delivery of two anticancer agents, doxorubicin and methotrexate, against MCF7 cancer cells. Several chemical characterizations were done for validation of prepared nanocarrier, and in vitro loading and release studies of drugs were performed with good encapsulation efficiency. Results: In vitro biological studies including hemolysis assay, erythrocytes sedimentation rate, red blood cells aggregation, cyto cellular internalization, and apoptosis evaluations were performed. Based on results, the developed nanocarrier has many advantages and capability for an efficient codelivery of DOX and MTX, which has a highly potent ability to kill cancer cells. Conclusion: All these results persuade us, this nanocarrier could be effectively used for cancer therapy of MCF7 breast cancer cells and is suitable for use in further animal studies in future investigations.