The aim of this study is to determine the cytotoxic and apoptotic effects of newly synthesized folic acid (FA) and luteolin (carboxylated luteolin (CL)) conjugated superparamagnetic nanoparticle (SPION@FA-PEG@CL) on folate receptor positive (FAR (+)) and negative (FAR (−)) cell lines in vitro. Cytotoxicity analysis by real-time cell analyzer system (RTCA) and apoptosis by TUNEL and Annexin assay, Caspase 3/7 activities, and Caspase 3/7 expression level were determined. According to RTCA results, SPION@FA-PEG@CL nanodrug showed higher cytotoxicity against U87, MCF-7, HeLa cell lines in comparison with L929 and A549 cell lines. TUNEL and Annexin assays indicated that SPION@FA-PEG@CL increased the apoptotic and necrotic cell ratio in FAR (+) cell. In L929 and A549 cells, are FAR (−) cell lines apoptotic and necrotic cell ratio was lower than FAR (+) cell lines (U87, MCF-7 and HeLa). Caspase 3 and 7 analyses also showed that SPION@FA-PEG@CL nanodrug have apoptotic and necrotic impact on cell lines, they were higher in FAR (+) cells than FAR (−) cells. According to results, synthesized SPION@FA-PEG@CL can be a novel nanodrug delivery system for in cancer chemotherapy.