The lateral flow assay (LFA) is one of the most successful analytical platforms for rapid on-site detection of target substances. This type of assay has been used in many rapid diagnoses, for example, pregnancy tests and infectious disease prevention. However, applications of LFAs for very small molecules remain a demanding challenge due to the problem of obtaining the corresponding binding partners to form sandwich complexes. In this paper, we report an affinity-switchable (AS) LFA (ASLFA) for the rapid and selective detection of hydrogen peroxide (H2O2), glucose, and ethanol in blood serum and urine samples. Unlike classical LFAs, which rely on the "always on" interaction between the antigen and the antibody, the working principle of ASLFA is based on the gold nanoparticle-conjugated AS biotin probe Au@H2O2-ASB, which can be activated by H2O2 for binding with the streptavidin (SA) protein. In the presence of glucose and ethanol, glucose oxidase and alcohol oxidase can react with the substrate to generate H2O2 and thereby activate Au@H2O2-ASB for binding with SA. Therefore, this ASLFA approach can be an alternative for classical glucose and ethanol detection methods in a wide variety of samples, where simple and rapid on-site detection is essential.