This study focuses on the synthesis and in vitro evaluation of magnetic nanocomposites (FeO@LDH) as methotrexate (MTX) delivery system for targeted anticancer therapy. In which, the FeO nanoparticles acted as magnetically responsive carriers; the coating layer of layered double hydroxide (LDH) was used as a storehouse for MTX. The prepared FeO@LDH nanocomposites exhibited a suitable size, good stability and magnetic responsibility (M 36.66emu/g); MTX successfully intercalated into the LDH of FeO@LDH at an entrapment rate (%) of 91.78% (the drug loading was 18.36%) by host-guest exchange process. Moreover, the release studies in vitro showed that the drug delivery system (FeO@LDH-MTX) had excellent pH-sensitivity, 84.94% of MTX was released within 48h at pH3.5 via the co-effect of dissolution of LDH layer and ion-exchange. WST-1 assays in cancer cells (MCF-7 and HepG2) and normal cells (HUVEC) demonstrated that FeO@LDH-MTX exhibited high anticancer activity while low toxicity to normal cells, and also the FeO@LDH composites were practically non-toxic. Thus, our results revealed that FeO@LDH-MTX would be a competitive candidate for targeted delivery and sustained and controlled release of MTX.