Binding affinity of biomolecular interactions can be directly extracted from measured surface plasmon resonance biosensor sensorgrams by fitting the data to the appropriate model equations. The conventional method for affinity estimation uses a series of analytes and buffers that are injected serially to a single immobilized ligand on the sensing surface, including a regeneration step between each injection, to generate information about the binding behavior. We present an alternative method to estimate the affinity using a single analyte concentration injected to multiple ligand densities in a microarray format. This parameter estimation method eliminates the need for multiple analyte injections and surface regeneration steps, which can be important for applications where there is limited analyte serum, fragile ligand-surface attachment, or the detection of multiple biomolecule interactions. The single analyte injection approach for binding affinity estimation has been demonstrated for two different interactant pairs, β2 microglobulin/anti-β2 microglobulin (β2M) and human IgG/Fab fragments of anti-human IgG (hIgG), where the ligands are printed in a microarray format. Quantitative comparisons between the estimated binding affinities measured with the conventional method are β2M: KD = 1.48 ± 0.28 nM and hIgG: KD = 12.6 ± 0.2 nM and for the single injection method are β2M: KD = 1.52 ± 0.22 nM and hIgG: KD = 12.5 ± 0.6 nM, which are in good agreement in both cases.