The residues of bisphenol A (BPA) in food packaging and water systems have a potential impact on human health; therefore, its analysis and detection have drawn scientists' attention. In this work, based on the change in fluorescence intensity resulting from the conformational switch of a berberine/BPA-aptamer system in the presence and absence of BPA, an ultra-sensitive fluorescence aptasensing system is proposed, in which BPA-aptamer is employed as the identification unit and berberine as the fluorescent probe. Various factors affecting the detection of BPA, including the concentration of the fluorescent probe, BPA-aptamer, BPA, pH, system stability time and other experimental conditions, were investigated in detail. Under the optimal experimental conditions, the fluorescence intensity of the sensing system of berberine/BPA-aptamer exhibited a good linear correlation with the BPA concentration in the range of 0-1300 μM with a LOD of 32 nM. The proposed fluorescent sensing system also exhibited excellent recoveries of 92.4-102.3% in tap water samples and showed good application prospects for the analysis and detection of BPA.