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Herein, we developed a novel truncation technique for aptamer sequences to fabricate highly sensitive aptasensors based on molecular docking and molecular dynamics simulations. The binding mechanism and energy composition of the aptamer/sulfaquinoxaline (SQX) complexes were investigated. We successfully obtained a new SQX-specific aptamer (SBA28-1: CCCTAGGGG) with high affinity (K_d = 27.36 nM) and high specificity determined using graphene oxide. This aptamer has a unique stem-loop structure that can bind to SQX. Then, we fabricated a fluorescence aptasensor based on SBA28-1, gold nanoparticles (AuNPs), and rhodamine B (RhoB) that presented a good linear range of 1.25–160 ng/mL and a limit of detection of 1.04 ng/mL. When used to analyze water samples, the aptasensor presented acceptable recovery rates of 93.1–100.1% and coefficients of variation (CVs) of 2.2–10.2%. In conclusion, the fluorescence aptasensor can accurately and sensitively detect SQX in water samples and has good application prospects.