Bovine serum albumin (BSA) containing buffers are the standard blocking buffer in biosensing, yet human serum is the intended application for most clinical sensors. However, the effect of human serum albumin (HSA) on binding assays remains underexplored. A simple and well-studied assay (human IgG/goat anti-human IgG) was investigated with a surface plasmon resonance (SPR) sensor to address this fundamental question in sensing. Calibrations were performed with buffers containing various concentrations of bovine or human serum albumin, as well as full and diluted bovine or IgG-depleted human serum. It was found that HSA or human serum, but not BSA or bovine serum, significantly affected the SPR shift and binding constants of the assay. Interestingly, large differences were also observed depending on whether the animal or human antibody was immobilized on the SPR chip for detection, highlighting that matrix protein/analyte/receptor interactions play a significant role in the response. We find that the interaction of soluble HSA with human IgG interferes with the recognition region, affecting the binding constant, and thus results obtained in BSA are not necessarily applicable to clinical samples or in vivo conditions. We also clearly demonstrate why a minimum dilution of 1 : 10 is often required in SPR assays to remove most background effects. Taken together, these results show that: (1) BSA does not affect the binding constant between antibodies and thus serves its purpose well when only surface blocking is intended, (2) HSA is an adequate surrogate for human serum in assay optimization, and (3) blocking buffers should be prepared with HSA in the optimization steps of assays to be translated to human blood or serum.