In this work, we report a novel method for the label-free detection of cyanotoxin molecules based on a direct assay utilizing a graphene-modified surface plasmon resonance (SPR) aptasensor. Molecular dynamic simulation of the aptamer’s interaction with cylindrospermopsin (CYN) reveals the strongest binding sites between C18–C26 pairs. To modify the SPR sensor, the wet transfer method of CVD monolayer graphene was used. For the first time, we report the use of graphene functionalized by an aptamer as a bioreceptor in conjunction with SPR for the detection of CYN. In a direct assay with an anti-CYN aptamer, we demonstrated a noticeable change in the optical signal in response to the concentrations far below the maximum tolerable level of 1 µg/L and high specificity.