Target recycling-oriented amplification has been widely applied for sensitive detection of DNA, RNA, and proteins due to its successful overcoming the inherent limitation of target-to-signal ratio of 1:1 in the traditional hybridization assay. Exonuclease III (Exo III) is usually used as the cleavage enzyme in the target recycling-oriented amplification because of its easy availability, high catalytic activity, and wide applicability. Even though Exo III is assumed to be double-stranded DNA (dsDNA) specific exonuclease in most literature, its cleavage of single-strand DNA (ssDNA) does occur, resulting in the target-independent degradation of probes. Herein, we design an intramolecular displacement probe with the capability of resistance to the nonspecific digestion of Exo III and fast hybridization kinetics. Through the substitution of 2-aminopurine for adenine in the intramolecular displacement probes, we develop a rapid and label-free approach to monitor Exo III-assisted target recycling amplification. We further demonstrate that this method can be used for the detection of DNA and proteins with excellent specificity and high sensitivity. Importantly, this method can be extended to rapid, label-free and multiplexed detection of various nucleic acids, proteins, and small molecules using different kinds of fluorescent nucleotide analogues and specific aptamers.