This work described a fluorometric and aptamer-based assay for aflatoxin B1 (AFB1). Aptamer-modified carbon dots (DNA-CDs) were first synthesized as fluorescence probes, then reacted with humic acid (HAs) which acted as quencher of the blue fluorescence of the CDs. It was found that HAs can readily adsorb ssDNA aptamers due to the presence of a rich surface chemistry (quinoidal units, aromatic rings and sugar moieties). This resulted in quenching of the fluorescence of the CDs (with excitation/emission peaks at 360/450 nm), probably due to π interactions. If the nanoprobe was reacted with AFB1, the DNA-CDs detached from the HAs and fluorescence was restored. Under optimized experimental conditions, the assay had a linear response in the 0.1-0.8 ng mL AFB1 concentration range, with a low limit of detection of 70 pg mL.