The emergence of nucleic acid isothermal amplification strategies based on soft nanoarchitectonics offers a new dimension to the traditional electrochemical technique, particularly because of its flexibility, high efficiency, and increased sensitivity for analytical applications. Various DNA/RNA isothermal amplification strategies have been developed for the design and fabrication of new electrochemical biosensors for efficient and important biomolecular detection. Herein, we provide an overview of recent efforts in this research field and the strategies for signal-amplified sensing systems, with their biological applications, current challenges and prospects in this promising new area.