We demonstrated that effects of serum matrix on molecular interactions between drugs and target proteins can be investigated in real time using magnetic bio-sensing techniques. A giant magneto-resistive (GMR) sensor was used on which target proteins were fixed and superparamagnetic nanoparticles (diameter: 50 nm) conjugated with drug were used in phosphate buffer, with and without serum. In this study, the following drug-protein pairs were investigated: quercetin and cAMP-dependent protein kinase A (PKA), Infliximab and tumor necrosis factor alpha (TNFα), and Bevacizumab and vascular endothelial growth factor (VEGF). For the quercetin and PKA pair, the time profile of the signal from the GMR sensor due to binding between quercetin and PKA clearly changed before and after the addition of serum. Moreover, it was revealed that not only the association process, but also the dissociation process was influenced by the addition of serum, suggesting that the quercetin and PKA complex may partially contain serum proteins, which affect the formation and stability of the complex. For antibody drugs, little effects of serum matrix were observed on both the association and dissociation processes. These clear differences may be attributed to the hydrophobic and electrostatic character of the drug molecule, target protein, and serum proteins. The real-time monitoring of molecular interactions in a biological matrix enabled by the GMR bio-sensing technique is a powerful tool to investigate such complicated molecular interactions. Understanding the molecular interactions that occur in a biological matrix is indispensable for determining the mechanism of action of the drugs and pharmacokinetics/pharmacodynamics inside the body. Additionally, this method can be applied for the analysis of the influence of any kind of third molecule that may have some interaction between two molecules, for example, an inhibitor drug against the interaction between two kinds of proteins.