A simple fluorescence method to quantitatively monitor malachite green (MG) was introduced based on the conformation switch effect of MG-aptamer (Apt) and the fluorochrome of SYBR Green I (SGI). SGI can embed in the stem-loop structure of Apt to produce fluorescence. However, the fluorescence will be quenched when MG is introduced because of the specific binding of Apt to MG. Under the optimal conditions, the fluorescence method displayed excellent linear relationship between the fluorescence quenching efficiencies and the MG concentrations ranging from 0.02 μmol L⁻¹ to 1.0 μmol L⁻¹. The detection limit of the method was 0.67 μg kg⁻¹ (3α/κ, n = 9) which is lower than the limit (1 μg kg⁻¹) specified in the national standard of China (GB/T 19857–2005) and the limit (2 μg kg⁻¹) specified by the European Food Safety Authority (EFSA, 2016). Excellent recoveries between 87.1% and 106.9% tested by spiking standards were achieved with RSD less than 6.4%. The data were confirmed by the method of HPLC, and the results proved that the established fluorescence method could be used for the rapid detection of MG in fish with high accuracy and sensitivity.