We develop for the first time an aptamer-mediated rolling circle amplification approach for label-free and sensitive detection of histone-modifying enzyme (HME) activity. This method can achieve femtomolar sensitivity for histone acetyltransferase Tip60 assay, which is the most sensitive HME assay reported so far. It can be further applied for inhibitor screening, enzyme kinetic analysis, and endogenous Tip60 measurement in cancer cells.