AIM: This study aims to prepare a nanocomposite (HPMC/5-FL@GO) from hydroxypropyl methylcellulose (HPMC) and graphene oxide (GO) as biocompatible materials. The nanocomposite enhances the drug activity of immobilized 5-fluorouracil (5-FU), decreasing the side effect of long-run treatment protocols with highly efficient drug-drug activity. METHOD AND RESULTS: Different samples were characterized by ATR-FTIR spectroscopy, X-ray diffraction, scanning electron microscopy coupled with energy dispersive X-ray analysis, transmission electron microscopy, thermogravimetric analysis, and dynamic light scattering along with cytotoxicity and anticancer study. A homogenous and compatible nanocomposite structure with a homogenous drug distribution was confirmed. Furthermore, the prepared nanocomposite has a low cytotoxicity effect against normal Vero cell lines compared with 5-FU. The antitumor activities of the same nanocomposite (20.4 and 74.3 μg mL on A549 and HepG-2) were lower than that of 5-FU (54.1 and 103 μg mL on A549 and HepG-2). CONCLUSION AND IMPLICATIONS: According to the attained results, the HPMC/5-FL@GO can apply in a biomedical application such as cancer therapy with the unique biocompatible to human cells.