Biocompatible magnetic poly (glycidyl methacrylate) microsphere is a novel nanocomposite with a myriad of promising bioapplications. Investigation of their characteristics by experimental analysis methods has also been carried out in the past. However, a survey of the magnetic anisotropy constant has not been mentioned and the influence of the poly (glycidyl methacrylate) polymer matrix on the Fe3O4 magnetite nanoparticles embedded inside has also not been discussed. Moreover, the accurate characterization of the magnetite nanoparticle size distribution remains challenging. In this paper, we present an effective approach was used to solve these problems. First of all, we combine both experiment and theory to estimate the effective magnetic anisotropy constant. Besides that, we implement an accurate method to determine magnetite nanoparticle size distribution in the magnetic poly (glycidyl methacrylate) microspheres composite nanomaterial.