Insulin, a peptide hormone secreted by pancreatic β cells, affects the development of diabetes and associated complications. Herein, we propose an electrochemical aptasensor for sensitive and selective detection of insulin using laser-scribed graphene electrodes (LSGEs). Before using disposable LSGEs, the development and proof-of-concept sensing experiments were firstly carried out on research-grade glassy carbon electrode (GCE). The aptasensor is based on using Exonuclease I (Exo I) that catalyses the hydrolysis of single-stranded aptamers attached to the electrode surface; however, the hydrolysis does not occur if the insulin is bound to the aptamer. Therefore, the unbound aptamers are cleaved by Exo I while insulin-bound aptamers remain on the electrode surface. In the next step, the gold nanoparticle - aptamer (AuNPs-Apt) probes are introduced to the electrode surface to form a 'sandwich' structure with the insulin on the surface-attached aptamer. The redox probe, methylene blue (MB), intercalates into the aptamers' guanine bases and the sandwich structure of AuNPs-Apt/insulin/surface-bound aptamer amplifies electrochemical signal from MBs. The signal can be well-correlated to the concentrations of insulin. A limit of detection of 22.7 fM was found for the LSGE-based sensors and 9.8 fM for GCE-based sensors used for comparison and initial sensor development. The results demonstrate successful fabrication of the single-use and sensitive LSGEs-based sensors for insulin detection.