Fumonisin B1 (FB1), a mycotoxin classified as group 2B hazard, is of high importance due to its abundance and occurrence in varied crops. Conventional methods for detection are sensitive and selective; however, they also convey disadvantages such as long assay times, expensive equipment and instrumentation, complex procedures, sample pretreatment and unfeasibility for on-site analysis. Therefore, there is a need for quick, simple and affordable quantification methods. On that note, aptamers (ssDNA) are a good alternative for designing specific and sensitive biosensing techniques. In this work, the assessment of the performance of two aptamers (40 and 96 nt) on the colorimetric quantification of FB1 was determined by conducting an aptamer-target incubation step, followed by the addition of gold nanoparticles (AuNPs) and NaCl. Although MgCl and Tris-HCl were, respectively, essential for aptamer 96 and 40 nt, the latter was not specific for FB1. Alternatively, the formation of Aptamer (96 nt)-FB1-AuNP conjugates in MgCl exhibited stabilization to NaCl-induced aggregation at increasing FB1 concentrations. The application of asymmetric flow field-flow fractionation (AF4) allowed their size separation and characterization by a multidetection system (UV-VIS, MALS and DLS online), with a reduction in the limit of detection from 0.002 µg/mL to 56 fg/mL.