The ideal way to assess aptamer affinity is when both aptamer and target are in a native state, without the unpredictable interference associated with labelling and surface immobilization. However, most current aptamer affinity assays need aptamer (or target) immobilization on surface and/or labelling. Ideally, such a solution-phase assay should also be high-throughput, in order to accelerate aptamer identification, binding site study, and engineering for various downstream applications. So far, only isothermal titration calorimetry (ITC) enables label-free solution-phase affinity measurements, but with low-throughput and the need of large amount of samples. Here, we report a solution-phase, label-free, colorimetric gold nanoparticle (AuNP)-based affinity assay (Nano-Affi) that addresses this need. Nano-Affi is based on kinetically-favoured, adsorbate charge-tuned aggregation of AuNPs, wherein positively-charged or near-neutral proteins induce instantaneous aggregation of negatively-charged AuNPs at the pH below or near the isoelectric point of the target protein. In contrast, protein-aptamer complexes possess a greater negative charge than free targets, and thus induce little or no aggregation of AuNPs due to electrostatic repulsion. The higher an aptamer's affinity for the protein, the less AuNP aggregation occurs. We demonstrate here that Nano-Affi enables the reliable aptamer screening and dissociation constant determination for diverse protein targets, as well as binding site identification, with readouts based on colour observation or absorbance or dynamic light scattering size measurements. Nano-Affi possesses sub-nanomolar sensitivity and can be performed with nanogram amounts of protein in less than half an hour with minimal training and minimal instrument requirements.