In this paper we show a high-throughput method to screen the kinetics and affinity constants of many biomolecular interactions simultaneously. During the preparation of the sensor chip, ligands were serially diluted and spotted in a 6 × 4 microarray on the sensor surface. A multi-analyte sample was injected and the real-time surface plasmon resonance (SPR) responses of all the 24 microarray spots were obtained using a commercial SPR imaging instrument. The multi responses of the association and dissociation processes obtained from a single analyte injection are sufficient to calculate the rates and affinity constants of the interactions between three interactant antigen-antibody pairs using a simple monophasic kinetic model. The method drastically reduces the measurement time and cost in the benefit of increased throughput.