Herein, a dual-signal output fluorescent aptamer sensor was constructed for the simultaneous detection of aflatoxin B (AFB) and ochratoxin A (OTA) using the specific recognition ability of aptamers and the programmability of DNA. A functional capture probe (cDNA) was designed with the black hole quenching motif BHQ1 labeled at the 5' end and biotin (bio) labeled at the 3' end. The fluorescent dye Cy3-labeled aflatoxin B1 aptamer (AFB-Apt) and the carboxyfluorescein FAM-labeled ochratoxin A aptamer (OTA-Apt) were used as two fluorescent probes. The cDNA is anchored to the quenching material gold nanoflowers (AuNFs) by the action of streptavidin (SA) and biotin. Its ends can be complementarily paired with two fluorescent probe bases to form a double-stranded structure. The fluorescence of Cy3 was quenched by AuNFs, and the fluorescence of FAM was quenched by BHQ1 through the fluorescence energy resonance transfer (FRET) effect, forming a fluorescence quenching system. Due to the high affinity of the target and the aptamer, the structure of the aptamer probe changes and detaches from the sensor when AFB and OTA are present, resulting in enhanced fluorescence. Under optimal conditions, the linear range of AFB was 0.1-100 ng/mL (R = 0.996), the limit of detection (LOD) was as low as 0.014 ng/mL, and the limit of quantification (LOQ) was 0.046 ng/mL. The linear range of OTA was 0.1-100 ng/mL (R = 0.995), the limit of detection (LOD) was as low as 0.027 ng/mL, and the limit of quantification (LOQ) was 0.089 ng/mL. The sensor had high accuracy in detecting both AFB and OTA in real sample analysis. The results of the t test show that there is no significant difference between the results of this study and the high-performance liquid phase (HPLC) method, indicating that the prepared sensor can be used as a potential platform for multiple mycotoxins detection.