The authors have incidentally found that the three tandem repeats of a 13-mer G-rich oligomer (with sequence 5'-TGG GAA GGG AGG G-3'; referred to as G3) can directly fold into a stable G3 trimer. The G3 trimer/hemin DNAzyme exhibits an about 3-fold higher peroxidase-mimicking activity compared to the conventional G3/hemin DNAzyme. Combining this finding with rolling circle amplification (RCA), a colorimetric assay was developed for sensitive and specific determination of microRNA. In this method, each cycle of RCA generates three catalytic units. This leads to a significant signal amplification of the RCA. Using let-7a as a model analyte, the colorimetric method (best performed at 420 nm) exhibits high sensitivity toward microRNA-let-7a with a 37 fM detection limit and an analytical range that covers 3 orders of magnitude. The method was applied to the determination of let-7a in some cell lysates. Graphical abstractThis G-triplex trimer-based rolling circle amplification (RCA) method can produce three catalytic units per RCA cycle, which can significantly improve the amplification efficiency of RCA.