Magnetic nanoparticles (MNPs) coated with Protein-G have been a model system to be used in different antibodies binding study. It is highly desirable to use a substrate-free biosensing system to detect antibodies binding in real-time. In this paper, we developed and applied a MNPs and search-coils integrated detection system, which is not only sensitive to the hydrodynamic volume of MNPs but also sensitive to the environment of MNPs, such as viscosity and temperature of the solution. We demonstrated that the viscosity effect influenced the amplitudes and phases of the 3rd (fH±2fL) and 5th (fH±4fL) harmonics for the mixed frequency testing scheme. The binding between antibodies and Protein-G on MNPs increased hydrodynamic volume of particles, as a result, it also changed the amplitudes and phases of harmonics, which are the object signals we need to analyze. We demonstrated that the viscosity of antibody solution is lower than that of MNP solution, and the antibody binding effect could be shielded by the viscosity effect to certain extent.