Signal amplification is vital in biosensor fabrication, especially for the sensitive analysis of target molecules. Rolling circle amplification (RCA) is an isothermal nucleic acid amplification technique. In a typical RCA process, a short DNA or RNA primer is extended under the catalysis of a polymerase to form a long single stranded DNA or RNA using a circular DNA template. In recent years, various approaches have been proposed to improve the RCA technique, so this nucleic acid amplification technique has become an attractive tool for biosensor fabrication, especially for the development of electrochemical biosensors with biomedical applications. Owing to the accumulation of RCA products on electrode surface, the electrochemical signal can be greatly amplified after an elaborated design so as to improve the detection sensitivity. Therefore, a variety of biomolecular targets such as genes, microRNAs, proteins, with ultra-low abundance have been successfully detected using the developed RCA-based electrochemical biosensors. In this review, we will summarize the improvement of the RCA technique and its application for the development of electrochemical biosensors with biomedical applications. We will also comment on the recent progress of RCA-based electrochemical biosensors, making an outlook on the trends of the related research fields in the future. (C) 2016 Elsevier B.V. All rights reserved.