Objective To develop a rapid fluorescence assay testing method for microRNA-721 (miRNA-721) detection using catalytic hairpin assembly for signal amplification without the need of enzymes. Methods A DNA hairpin probe H1 labeled with 5-carboxyfluorescein (FAM) was modified on the surface of gold nanoparticle (AuNP) to form probe AuNP-H1, and the fluorescence of H1 was quenched by AuNP. When the target miRNA was introduced, it caused the fluorescein modified in H1 on the AuNP to move away from the AuNP and emit fluorescence. This was followed by cyclic self-assembly between H1 and H2, and the target cycle was used to amplify the fluorescence signal. The fluorescence intensity of the system was measured at 480 nm excitation wavelength after co-reacting with the probe AuNP-H1, probe H2 and different concentrations of miRNA-721. Results When biomarker of acute myocarditis miRNA-721 was used as a model target, 20 μL of probe AuNP-H1 and 50 μL of 3 μmol/L probe H2 (annealing buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, 5 mmol/L MgCl₂, pH 7.4) were co-reacted with 50 μL of miRNA-721 at various concentrations ranging from 0.1 to 5 μmol/L at 30℃ for 20 min. The change in relative fluorescence intensity at 520 nm (∆_F＝F－F₀) had a good linear relationship with the concentration of miRNA-721 (C), and the fitted linear equation was ∆_F＝29 232×lgC－52 435 (R²＝0.991 0). The detection limit of this fluorescence method was 1.23 nmol/L. The recovery rate in normal human serum ranged from 92.71% to 104.02%. A comprehensive miRNA analysis could be completed in just 20 min. Conclusion The present method can be applied to detect miRNA-721 in biological samples, which providing a rapid diagnostic tool for acute myocarditis.