This study presents a one-step, green synthesis method for copper single-atom nanozymes (CuSANs) on laser-scribed graphene (LSG) for electrochemical sensing of hydrogen peroxide (H₂O₂). The CuSANs are synthesized using a CO₂ laser to simultaneously generate LSG support and anchor copper atoms on a polyimide sheet. The CuSANs exhibit a surface metal loading of 1.47% ± 0.16%, confirmed by high-angle-annular dark-field scanning transmission electron microscopy and X-ray photoelectron spectroscopy. The CuSANs are used as a working electrode in an electrochemical sensor for H₂O₂ detection, achieving a detection limit of 2.40 μM. The sensor demonstrates high sensitivity, with a sensitivity of 130.0 μA mM⁻¹ cm⁻², making it suitable for oxidative stress assessment. The CuSANs show enhanced catalytic activity for H₂O₂ reduction, with a threefold increase in current compared to bare LSG. The sensor is selective for H₂O₂, showing minimal interference from common interferants like dopamine, glucose, and uric acid. The sensor is also reproducible, with a relative standard deviation of 16.25%. The CuSANs are stable over 28 days, with a shelf-life stability of 87.87%. The method is simple, fast, and environmentally friendly, offering a promising approach for the development of point-of-care devices for H₂O₂ detection in blood. The study highlights the potential of CuSANs as a robust and efficient alternative to traditional enzymes for electrochemical sensing applications.This study presents a one-step, green synthesis method for copper single-atom nanozymes (CuSANs) on laser-scribed graphene (LSG) for electrochemical sensing of hydrogen peroxide (H₂O₂). The CuSANs are synthesized using a CO₂ laser to simultaneously generate LSG support and anchor copper atoms on a polyimide sheet. The CuSANs exhibit a surface metal loading of 1.47% ± 0.16%, confirmed by high-angle-annular dark-field scanning transmission electron microscopy and X-ray photoelectron spectroscopy. The CuSANs are used as a working electrode in an electrochemical sensor for H₂O₂ detection, achieving a detection limit of 2.40 μM. The sensor demonstrates high sensitivity, with a sensitivity of 130.0 μA mM⁻¹ cm⁻², making it suitable for oxidative stress assessment. The CuSANs show enhanced catalytic activity for H₂O₂ reduction, with a threefold increase in current compared to bare LSG. The sensor is selective for H₂O₂, showing minimal interference from common interferants like dopamine, glucose, and uric acid. The sensor is also reproducible, with a relative standard deviation of 16.25%. The CuSANs are stable over 28 days, with a shelf-life stability of 87.87%. The method is simple, fast, and environmentally friendly, offering a promising approach for the development of point-of-care devices for H₂O₂ detection in blood. The study highlights the potential of CuSANs as a robust and efficient alternative to traditional enzymes for electrochemical sensing applications.