To ensure the safety of the natural ecosystem, it is critical to develop portable, easy-to-operate and environmentally friendly quantitative analytical electrodes for the determination of microcystin-LR (MC-LR) in environmental water. Herein, a simple, mask-free, and one-step method based on laser-induced graphene (LIG) technology is designed for the fabrication of (3D) graphene electrodes integrated with gold nanoparticles (AuNPs). The 3-electrode system with AuNPs-modified graphene was prepared directly by overlaying gold leaf on the polyimide (PI) film followed by patterned CO2 laser irradiation. The reference electrode region was then uniformly coated with Ag/AgCl paste, and the working electrode was functionalized with the aptamer through Au-S self-assembly and bonding to obtain a label-free MC-LR aptasensor. The developed MC-LR aptasensor has a detection limit of 0.053 pM and a linear range from 0.1 pM to 10 nM. Furthermore, the fabricated aptasensor presents good stability, reproducibility, specificity and detection in real samples. These sensors show great promise as a low-cost, simple and portable approve for label-free detection MC-LR in environmental samples.