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Molecular recognition (e.g., antigen-antibody, DNA-DNA, and streptavidin-biotin) is a generic, yet highly versatile and powerful strategy employed in enzyme-catalyzed signal amplification process. However, this approach is not applicable to metals, anions, and small reactive species (e.g., O2- and F-), as these molecules are too small to bind effectively to the macromolecules. In this paper, we demonstrate an enzyme-catalyzed signal amplification approach based on the controlled binding between streptavidin and target activated affinity-switchable biotin (ASB) probes, for the detection of O2- and F-, using electrochemical and fluorescent detection techniques. The underlying rationale behind this design is that, while the ASB probe would not bind with the streptavidin-enzyme conjugate due to its low binding affinity with streptavidin, in the presence of the target analyte, the ASB probe on the immobilized surface will be activated to form biotin, which can then bind with the enzyme-tagged streptavidin to initiate signal amplification process. This versatile approach can also be applied in the imaging of endogenously secreted O2- along the plasma membrane of living cells using streptavidin conjugated with multiple fluorescent dye reporters. We believe that this ASB probe strategy will be useful for a wide range of applications, such as in basic biological research and medical diagnoses, where highly specific signal enhancement is required.