A label-free electrochemical aptasensor was fabricated to sensitively determine malachite green (MG) based on Au nanoparticles/graphene quantum dots-tungsten disulfide nanosheet composite film modified glassy carbon electrode (AuNPs/GQDs-WS₂/GCE). A facial strategy for the self-assembly of graphene quantum dots (GQDs) on tungsten disulfide nanosheets (WS₂) was developed to fabricate 0D/2D nanocomposites. As-prepared GQDs-WS₂ hybrids exhibited significantly enhanced electrocatalytic properties, and were first used as electroactive materials to construct electrochemical aptasensor. The AuNPs/GQDs-WS₂/GCE was prepared through depositing Au nanoparticles on the surface of the GQDs-WS₂ film, which acted as the electrochemical sensing matrix to covalently immobilize the aptamers of MG via the Au–S bond. In this label-free proposal, the aptasensor was applied to detect MG by monitoring voltammetric signal resulted from electrochemical oxidation of the MG captured by the aptamer. Under the optimized conditions, the aptasensor showed a wide linear range from 0.01 to 10 μM for MG detection with a low detection limit of 3.38 nM (S/N = 3). The method was applied to determination of MG in spiked fish samples and gave satisfactory results.