The article presents a novel, environmentally friendly, and single-step method for the synthesis of copper single-atom nanozymes (CuSANs) on laser-scribed graphene (LSG) using a CO2 laser. The CuSANs are characterized by high surface metal loading (1.47% ± 0.16%) and lack of aggregation, demonstrating uniform dispersion of atomic Cu on the LSG surface. The electrochemical properties of CuSANs are evaluated, showing enhanced sensitivity to hydrogen peroxide (H2O2) with a detection limit of 2.40 μM and a linear range of 3.0 μM to 1.0 mM. The CuSANs exhibit superior performance compared to existing H2O2 sensors, making them suitable for point-of-care devices and applications in oxidative stress assessment. The study highlights the potential of CuSANs in biosensing and biomedical applications.The article presents a novel, environmentally friendly, and single-step method for the synthesis of copper single-atom nanozymes (CuSANs) on laser-scribed graphene (LSG) using a CO2 laser. The CuSANs are characterized by high surface metal loading (1.47% ± 0.16%) and lack of aggregation, demonstrating uniform dispersion of atomic Cu on the LSG surface. The electrochemical properties of CuSANs are evaluated, showing enhanced sensitivity to hydrogen peroxide (H2O2) with a detection limit of 2.40 μM and a linear range of 3.0 μM to 1.0 mM. The CuSANs exhibit superior performance compared to existing H2O2 sensors, making them suitable for point-of-care devices and applications in oxidative stress assessment. The study highlights the potential of CuSANs in biosensing and biomedical applications.