Rolling circle amplification is a widely used biosensing technique. Although various secondary structures have been employed in RCA, their effects on RCA efficiency have seldom been reported. Here, we find that stems in circular templates can strongly inhibit RCA, and the primer-stem distance is responsible for the inhibition. Based on the results, we propose an initiation inhibition mechanism and present a design principle for a general RCA assay. Inspired by this mechanism, we further propose a new nucleic acid detection method. The results verify that this method can increase RCA detection sensitivity according to the target recycling principle. Besides DNA detection, it can also achieve single mismatch discrimination of miRNA detection after optimization. This method also shows convenient visualization detection. The initiation inhibition of RCA could be helpful for RCA applications as promising detection techniques.