There is an urgent need for reliable biosensors to detect nucleic acid of interest in clinical samples. We propose that the accuracy of the present nucleic acid-sensing method can be advanced by avoiding false-positive identifications derived from nonspecific interactions (e.g., nonspecific binding, probe degradation). The challenge is to exploit biosensors that can distinguish false-positive from true-positive samples in nucleic acid screening. In the present study, by learning from the enzymatic cycle in nature, we raise an allostery tool displaying invertible positive/negative cooperativity for reversible or cyclic activity control of the biosensing probe. We demonstrate that the silencing and regeneration of a positive (or negative) allosteric effector can be carried out through toehold displacement or an enzymatic reaction. We, thus, have developed several dynamic biosensors that can repeatedly measure a single nucleic acid sample. The ability to distinguish a false-positive from a true-positive signal is ascribed to the nonspecific interaction presenting equivalent signal variations, while the specific target binding exhibits diverse signal variations according to repeated measurements. Given its precise identification, such consequent dynamic biosensors offer exciting opportunities in physiological and pathological diagnosis.