Sterigmatocystin (STC) is a toxic fungal secondary metabolite recognized by the FAO and WHO as a genotoxic and carcinogenic substance. STC contaminates several foods and feed commodities, posing a health risk to humans. The present study proposes to develop a graphene oxide-mediated aptasensor platform for the one-step detection of STC. In this study, DNA aptamers were generated against STC by using a target immobilization-free graphene oxide (GO)-SELEX protocol. The champion aptamers were subjected to in silico maturation using a genetic algorithm to improve binding affinity. Further, MSA-C6 and STC interactions were characterized by MD simulation, bio-layer interferometry (K_D 27.9 nM) and flow cytometry. GO was immobilized on a polypropylene surface and functionalized with FAM labelled MSA-C6 to develop a simple one-step fluorescence turn-on aptasensor. The linear detection range of the aptasensor was found to be 80–720 ppb with LOD 23.56 ± 4.93 ppb and LOQ 132.43 ± 3.25 ppb. Insignificant interference of salts and detergents as well as negligible cross-reactivity with other structurally similar mycotoxins were observed. Recovery studies in simulated contaminated samples indicated appreciable recoveries (71–89%) using aptasensing assay. The results of the study indicate the successful development of a simple one-step detection platform for STC, useful for the measurement and monitoring of samples for the presence of STC. It also reports a high-affinity aptamer, which can be exploited in other sensing platforms.