New developments in microfluidic chip technology enable the construction of chemical spectrum analyzers that can probe the binding interactions between chemical entities. In this paper we report the implementation of a microfluidic chip suitable for Fourier transform measurements of biochemical interactions. The chip consists of a chemical signal generator, a flow cell and a binding sensor surface. The microfluidic signal generator produces a periodic stream of protein plugs in solution flowing at constant velocity through the cell. This flow produces periodic association and dissociation cycles of the protein to a functionalized gold sensing surface placed inside the cell. The sensor activity corresponding to the phasor response of the chemical interaction at the excitation frequency is measured optically using surface plasmon resonance (SPR) imaging. We demonstrated the feasibility of the technique using a model system of carbonic anhydrase-II (CA-II) and immobilized 4-(2-Aminoethyl) benzenesulfonamide (ABS) ligand. The observed transfer function showed a dominant pole at 10.2 mHz corresponding to association and dissociation constants of 4.8 × 10(3) M(-1)·s(-1), and 3.5 × 10(-2) s(-1) respectively.