In this study, a stable carboxylated luteolin (CL)-functionalized superparamagnetic iron oxide nanoparticle (SPION) as a potential drug carrier for in vitro analysis of L929 (mouse fibroblast), U87 (glioblastoma (brain cancer)), MCF-7 (breast cancer), HeLa (cervix cancer), and A549 (human lung cancer) cell lines has been synthesized. Thermal decomposition and Stober methods were used to prepare 3-aminopropyl triethoxysilane-capped SPIONs respectively. Carboxylated polyethylene glycol (PEG-COOH), folic acid (FA), and CL were conjugated on the surface via a carboxylic/amine group using the nanoprecipitation method respectively. Internalization of CL-functionalized SPION and the release of luteolin from it has been studied using Prussian blue staining and spectrophotometry respectively. The cytotoxicity of CL-functionalized SPION on cell lines was tested by MTT assay. Internalization of product by HeLa, MCF-7, and U87 was higher than A549 and L929 cells. It was observed that luteolin release increased in an acidic environment (pH 5.4). A newly synthesized SPION-FA-PEG in all concentrations (except 500 mu g/mL) did not show notable toxicity against L929, U87, MCF-7, HeLa, and A549. However, the product in all used concentrations decreased cell viability at the 24th and 48th hours. This study confirmed that the product has a potential to be used as an anti-cancer CL-functionalized SPION for targeted drug delivery.