In this paper, a padlock probe-based exponential rolling circle amplification (P-ERCA) assay is developed for highly specific and sensitive detection of microRNA (miRNA). The padlock probe is composed of a hybridization sequence to miRNA and a nicking site for nicking endonuclease. Using the miRNA as a template, specific ligation to the padlock probe and linear rolling circle reaction (LRCA) are achieved under isothermal conditions. After multiple nicking reactions, many copies of short DNA products are successively produced and then used as triggers in next circle amplification. Thus, a small amount of miRNAs are converted to a large number of triggers to initiate the rolling circle amplification reaction, and circular exponential signal amplification is achieved. This padlock probe-based exponential rolling circle amplification assay exhibits a remarkable sensitivity of 0.24 zmol using optimized sequences of the padlock probe. The target-dependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to single-nucleotide difference discrimination between miRNA family members. The miRNA analysis in human lung cells was performed with this method. The result indicates this highly sensitive P-ERCA strategy will become a promising miRNA quantification method in early clinical diagnostics.