Fumonisin B1, a mycotoxin commonly produced by Fusarium verticillioides and classified as group 2B hazard, has been quantified in a variety of food products. After the first reported aptamer (96 nt ss-DNA) for the highly specific molecular recognition of FB1, only 28 aptamer-based biosensors have been published, of which 50% quantified fluorescent signals. A critical point, yet commonly overlooked during the design of aptasensors, is the selection of the binding buffer. In this work, a simplified colorimetric assay was designed by incubating a folded aptamer with FB1, and the subsequent addition of gold nanoparticles (AuNP). The change in the aggregation profile of AuNP by a 40 nt aptamer or a 96 nt aptamer, was tested after addition of the target molecule under different buffer conditions. The incubation with Tris-HCl and MgCl₂, exhibited the most favorable performances from the 40-mer and 96-mer, respectively; however contrary to previous publications, the short sequence was not specific to FB1 as it also portrayed signal suppression with other mycotoxins. On the other hand, the assay developed with the longest aptamer was specific to FB1 and comparable to other sensitive aptasensors with a limit of detection of 3 ng/mL (A_650/520 ratio) and 2 ng/mL (absolute peak area). Contrary to other mechanisms, particle stabilization was triggered by an increasing target concentration. Additionally, the application of more complex methods such as asymmetric flow field-flow fractionation (AF4) allowed the analysis of diverse signals (peak area (λ), refractive index, average diameter), to increase the sensitivity up to the pg/mL level. To our knowledge, this is the first colorimetric study reporting the sole application of an unmodified aptamer for the detection of FB1, thus avoiding the application of complementary sequences and allowing the formation of a complex AuNP-aptamer-FB1 caused by MgCl₂.