Specific absorption rate (SAR) of magnetic iron oxide (Fe3O4) nanoparticles (NPs) is an important property in hyperthermia applications. In this work, the dependence of magnetic anisotropy (K) on concentration of Fe3O4 NPs has been investigated using the Monte Carlo simulations. The results showed that the K value increases with the NPs concentration which helps to clarify the dual behavior of both increase and decrease of SAR value with concentration based on the Linear Response Theory (LRT). The theoretical results explained the influence of concentration on SAR based on the relationship between magnetic anisotropy and inter-particle distance. Furthermore, Fe3O4 NPs of size range from 7 to 17 nm have been synthesized with high magnetization saturation (65.1–68.1 emu/g) and their superparamagnetic behaviors have been determined. The experimental results indicated that the SAR value could increase with the concentration and has a bell shape at a specific size of MNPs, which are in good agreement with the theoretical simulation. All theoretical and experimental study of SAR recognized the important role of magnetic anisotropy to enhance SAR values. Especially, the results showed that there exists an optimal concentration at 15 mg/ml for 17 nm Fe3O4 NPs that maximized SAR value.