Graphene oxide (GO)-based aptasensors are currently one of the most popular sensing platforms for the simple and rapid detection of various targets. Unfortunately, the GO-based aptasensors with long aptamer strands typically show unsatisfactory performance resulting from insignificant structural transformations upon target binding. We report herein the utilization of an aptamer-truncating strategy to combat such a challenge. Taking a pre-selected anti-aflatoxin B1 (AFB1) aptamer (P-AFB1-50) as a trial system, we sequentially remove the extraneous nucleotides within the aptamer by means of circular dichroism (CD) spectroscopy and binding affinity analysis. Particularly, the ratio of the quenching constants between the GO sheets and the truncated aptamers (labelled with fluorophores) in the absence and presence of the target was determined for each of the truncated aptamers to evaluate the optimal sequence. As a result, the truncated aptamer comprising 40 nucleotides was confirmed to show the highest FL output and the best detection limit upon conjugation with GO sheets. More importantly, we demonstrated that this truncating strategy is versatile, i.e., it can be easily extended to other aptamer systems (anti-ochratoxin A (OTA) aptamer, P-OTA-61, as an example) for extraneous nucleotide identification. Impressively, the two optimal truncated aptamers can work together on GO sheets to achieve a simultaneous detection of two different mycotoxins (i.e., AFB1 and OTA) in one single test. Essentially, this research opens a new avenue for the design and testing of aptamer-/GO-based-sensing platforms for rapid, low-cost and multiplex quantification of analytical targets of interest.