The isothermal enzyme‐free nucleic acid amplification method plays an indispensable role in biosensing by virtue of its simple, robust, and highly efficient properties without the assistance of temperature cycling or/and enzymatic biocatalysis. Until to now, enzyme‐free nucleic acid amplification has been extensively utilized for biological assays and has achieved the highly sensitive detection of various biological targets, including DNAs, RNAs, small molecules, proteins, and even cells. In this review, the mechanisms of entropy‐driven reaction, hybridization chain reaction, catalytic hairpin assembly and DNAzyme were concisely described and their recent application as biosensors was comprehensively summarized. Furthermore, the current problems and the developments of these DNA circuits were also discussed.