The ultrasensitive DNA methyltransferase (Dam MTase) assay is of high significance for biomedical research and clinical diagnosis because of its profound effect on gene regulation. However, detection sensitivity is still limited by shortcomings, including photobleaching and weak signal intensities of conventional fluorophores at low concentrations. Plasmonic nanostructures with ultrastrong electromagnetic fields and fluorescence enhancement capability that can overcome these intrinsic defects hold great potential for ultrasensitive bioanalysis. Herein, a silica-coated gold nanostars (Au NSTs@SiO2)-based plasmon-enhanced fluorescence (PEF) probe with 20 "hot spots" was developed for ultrasensitive detection of Dam MTase. Here, the Dam Mtase assay was achieved by detecting the byproduct PPi of the rolling circle amplification reaction. It is worth noting that, benefiting from the excellent fluorescence enhancement capability of Au NSTs originating from their 20 "hot spots", the detection limit of Dam Mtase was reduced by nearly 105 times. Moreover, the proposed Au NST-based PEF probe enabled versatile evaluation of Dam MTase inhibitors as well as endogenous Dam MTase detection in GW5100 and JM110 Escherichia coli cell lysates, demonstrating its potential in biomedical analysis.