The study of biomolecular interactions is of basic importance in understanding processes of molecular recognition and biological function. An area of recent development is the use of biosensors to monitor interactions in real time. In this work, Real-time BIA (Biomolecular Interaction Analysis) from Biacore AB, that uses the optical phenomenon surface plasmon resonance (SPR), was a useful technique to investigate the interaction between enzyme and protease inhibitors. The enzymes (trypsin and chymotrypsin) were coupled to a carboxymethyldextran CM5 sensor chip by amine coupling. The novel protease inhibitor isoforms, denoted as ApTI A, ApTIB and ApTIC, were purified from the crude extract of Acacia plumosa seeds. The isoinhibitors are 20 kDa protein, constituted of two polypeptide chains about 16 and 4 kDa linked by disulfide bond. The stoichiometry observed for the complex were 1:1 with both enzyme. The binding assay was performed with ApTI isoforms in the range of 1-128 nM. Association (K_A) and dissociation (K_D) constants of each assay, obtained with BIAevaluation Version 4.1 software, indicated the high affinity of these inhibitors for the enzymes site (around 2.6x10⁹ M^-1 for trypsin and 1.4x10⁸ M^-1 for chymotrypsin) and high stability of the resultant complex (around 0.5 nM for trypsin and 6 nM for chymotrypsin), showing stronger interactions with trypsin than chymotrypsin. The same assay is also being investigated with other serine proteases (elastase and kallikrein). By analyzing the values of interaction (K_A and K_D) and inhibition constants (K_i), we can conclude the binding and enzyme inhibiton were closely correlated, where a direct binding assay performed with biosensor technology can provide a rapid and useful alternative to investigate more elaborate inhibition experiments and may also be useful for characterization of other macromolecular interactions.