We presented a new aptasensor for mycotoxins, which was based on multiplexed fluorescence resonance energy transfer (FRET) between multicolor upconversion fluorescent nanoparticles (UCNPs) as donors and graphene oxide (GO) as the entire and effective acceptor. BaY(0.78)F(5):Yb(0.2), Er(0.02) and BaY(0.78)F(5):Yb(0.7), Tm(0.02) upconversion nanoparticles were synthesized and functionalized, respectively, with immobilized ochratoxin A (OTA)-aptamers and fumonisin B(1) (FB(1))-aptamers. On the basis of the strong π-π stacking effect between the nucleobases of the aptamers and the sp(2) atoms of GO, the aptamer modified-UCNPs can be brought in close proximity to the GO surface. The strong upconversion fluorescence both of BaY(0.78)F(5):Yb(0.2), Er(0.02) and BaY(0.78)F(5):Yb(0.2), Tm(0.02) can be completely quenched by the GO, because of a good overlap between the fluorescence emission of multicolor UCNPs and the absorption spectrum of GO. In contrast, in the presence of OTA and FB(1), the aptamers preferred to bind to their corresponding mycotoxins, which led to changes in the formation of aptamers, and therefore, aptamer modified-UCNPs were far away from the GO surface. Our study results showed that the fluorescence intensity of BaYF(5):Yb Er and BaYF(5):Yb Tm were related to the concentration of OTA and FB(1). We therefore developed a sensitive and simple platform for the simultaneous detection of OTA and FB(1) with multicolor UCNPs and GO as the FRET pair. The aptasensor provided a linear range from 0.05 to 100 ng·mL(-1) for OTA and 0.1 to 500 ng·mL(-1) for FB(1); the detection limit of OTA was 0.02 ng·mL(-1) and FB(1) was 0.1 ng·mL(-1). As a practical application, the aptasensor was used to monitor OTA and FB(1) level in naturally contaminated maize samples with the results consistent with that of a classic ELISA method. More importantly, the novel multiplexed FRET was established for the first time based on multiplexed energy donors to the entire energy acceptor; this work was expected to open up a new field of FRET system applications for various targets.