Fumonisin B1 (FB1) and ochratoxin A (OTA) are agriculturally important mycotoxins that have implications in human and animal health. Aptamers are short single-stranded oligonucleotides that can be used as molecular recognition elements for the sensitive and selective detection of mycotoxins. Aptamers for FB1 and OTA were screened using a novel DNase I footprinting assay to characterize their structure and in- solution affinity. Two minimal binding aptamers for FB1 were identified that retain high binding affinity in solution and to a bound target. Progress was made towards fluorescence turn-on and turn-off sensors for FB1, utilizing semiconductor quantum dots and fluorescein in conjunction with metallic- and carbon-based quenchers. A fluorescence nano-aptasensor was developed as a simple paper test, with the capacity to detect OTA with a limit of detection between 10 – 100 nM. Rapid, robust aptasensors for FB1 and OTA have promising applications for in-field screening of mycotoxin residues on crops.