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Graphene oxide (GO) is one of the promising two-dimensional nanomaterials for the development of fluorescent aptasensors. Its limitations are related to the inevitable negative effect of GO on the fluorescence. Here this challenge was explored and solved by ssDNA adsorption on GO and centrifugal separation of GO from the ensembled solution. On this basis, a fluorescent aptasensor was constructed for the detection of staphylococcal enterotoxin A (SEA), in which GO assisted the separation of free aptamers to achieve cDNA-induced structure-switching from SEA/aptamer complex to dsDNA, and GO was added again to separate free cDNA and reduce the background signal. SYBR Green I was inserted into dsDNA as the fluorescent signal to achieve the quantitative analysis. It displayed a wide linear range (1–8000 ng/mL), low detection limit (0.899 ng/mL), and satisfactory application in milk and milk powder. Notably, the method was low-cost, general, and instructive for other two-dimensional nanomaterials. [Display omitted] • A label-free, low-background fluorescent aptasensor for staphylococcal enterotoxin A. • Graphene oxide-assisted ssDNA separation to reduce the background and interference. • The cDNA-induced structure-switching formed dsDNA and amplified the signal. • The method was low-cost, general, and instructive for two-dimensional nanomaterials. [ABSTRACT FROM AUTHOR]