Introduction: Graphene oxide (GO) sheets are carbon-networking nanomaterials offering excellent potential for drug delivery platforms due to hydrophobic interactions and high drug-loading efficiency. Superparamagnetic iron oxide nanoparticles can be used in certain applications such as cell labeling, drug delivery, targeting, magnetic resonance imaging and hyperthermia. Due to the larger surface to volume ratio for unmodified iron oxide nanoparticles, they usually possess high surface energy leading to particle aggregation. The magnetic nanoparticles could induce heat energy in an alternating magnetic field (AMF). The absorption of radiofrequency energy by biological tissue results in higher tissue temperatures as a function of time, which eventually causes cell death due to hyperthermia. Mensuration of the temperatures inside the tumors and in normal tissues within AMF demonstrated that this treatment could induce tumor-specific hyperthermia. Materials and Methods: CT26 colon cancer cell line was cultured in vitro as a monolayer. The monolayer culture was treated with of 5-FU, 5-FU- MGO nanoparticles, and MGO nanoparticles for 24 hours. then the flasks were located at the centre of the RF coil where the alternative magnetic field strength had reached its maximum value (40 kA/m) and was exposed to the AMF (forward power, 70 W), for 12 min (43°C) without MGO nanoparticles and 4 min (43°C) with MGO nanoparticles. During the AMF exposure, the temperature increase was monitored using a T-type thermocouple (copperconstantan, 0.1 mm diameter) linked to a digital thermometer (Lutron thermometer TM-917, Taiwan) every 1 min for 15 min with ± 0.1°C accuracy. Results: Increase in the temperature was measured. The results revealed that the temperature increase of the culture depends on the nanoparticles concentrations. The results also indicated that the temperature of cells exposed to AMF (13.56 MHz) at a forward power of 70 W reached to 43°C after 12 min, whereas the combination of accumulated 50 μg/ml 5-FU- MGO nanoparticles and RF hyperthermia was 3 min and for 25 and 5 μg/ml, it was calculated as 4 and 9 min, respectively. The significant difference was observed between the 5-FU, 5- FU- MGO nanoparticles, and MGO nanoparticles in RF hyperthermia at power of 70 W (P < 0.05). This suggests that MGO nanoparticles act as effective thermal agents in AMF hyperthermia. Conclusion: On the basis of this study is According to the temperature-time curves at different intensities, the rate of temperature rise was very sharp during the first few minutes of AMF exposure; however, it then reached a near equilibrium because cell heat transfer parameters (conduction and convection) started to perform, The radiofrequency heats cells with a steep gradient pending the initial stage of exposure.