We developed and validated a biosensor-based surface plasmon resonance (SPR) technology for the biological quantification and quality control (QC) of pharmaceutical proteins using reference materials as the standard. The surface of the receptors was made homogeneous by covalently immobilizing the receptors onto Au-membrane microchips for use as biosensors for reliably detecting the activity of drug proteins. This assay used only limited amounts of ligands and no additional detection agents. The products were determined to have binding capacity equivalent to that of the reference materials and to exhibit a recovery range of 88.4–115.0%. The binding of analytes to the specific ligand is concentration dependent and parallel. CTLA-4 fusion proteins were quantitatively detectable at concentrations as low as 125 ng/mL. The intra-assay precision was in the range of 1.07–7.27%, and the inter-lot precision was 13.03%. These data proved the usefulness of improved biosensor-based assays in biological quantification and QC of pharmaceutical proteins. This approach is an alternative to traditional assays and offers a potentially significant advantage in that the microchip can be regenerated thus enabling multiple analyses to be performed with a single sensor.