Binding affinity characterization is of great importance for aptamer screening because dissociation constant(Kd)value is a significant parameter for evaluating molecular interaction.However,conventional screening methods often require sophisticated scientific equipments to give an accurate readout.Here,we present a portable platform,Afi-Chip,as an equipment-free,fast,low-cost and universal platform for evaluation of the aptamer binding affinity.The Afi-Chip displays a distance readout for describing the amount of binding aptamer based on the reaction of enzyme catalyzing the decomposition of H2O2 for gas generation.Taking the advantage of translating recognition signal to distance signal and realizing the regents mixing and quantitative readout on the glass chip,we successfully monitored the aptamer evolution process and characterized the binding affinity of aptamers against multiple types of targets,including small molecular glucose,cancer biomarker protein EpCAM,and tumor cell SW620.We also applied it for rapid characterization of the noncovalent interactions between anti-HCG and HCG to demonstrate the generality of Afi-Chip approach for molecular interaction study.All of the Kd values are similar to those reported in literature or obtained by sophisticate instruments such as flow cytometer.Afi-Chip offers a new approach for equipment-free investigation of molecular interactions,such as aptamer identification,ligand selection monitoring,drug screening and inhibitor screening.