BACKGROUND: Hydrogen peroxide (H₂O₂) plays an essential role in different reactions and mechanisms. Therefore, simple and sensitive detection of H₂O₂ becomes very important for both medical and environmental applications. RESULTS: Two-step immobilization of horseradish peroxidase (HRP) on acid-treated single-walled carbon nanotubes (cut-SWCNTs) was reported for the sensitive detection of H₂O₂ at physiological conditions. Cut-SWCNTs were first synthesized using acid treatment and then coated onto screen-printed electrodes (SPEs) for electrochemical optimization tests. Electrochemical characterization of cut-SWCNTs coated CSPEs showed a linear relationship between current and increasing material loading. Also, the films demonstrated good reproducibility and stability. Immobilization of HRP was then achieved successfully using pyrene crosslinking chemistry on cut-SWCNT coated SPEs. The detection of H₂O₂ was achieved with HRP/cut-SWCNT/SPE electrode configuration using voltammetry technique. The constructed biosensor has sensitivity and apparent Michaelis–Menten constant (K_m) values of 34.1 μA L mmol^–1 cm⁻² and 0.47 mmol L^–1, respectively within the linear range of 20 to 600 μmol L^–1 H₂O₂ concentration. The biosensor also showed a good degree of reproducibility and low cross-contamination for tested interferences. CONCLUSION: H₂O₂ biosensor was successfully developed using cut-SWCNTs and HRP enzyme establishing successful direct electrochemical communication between the enzyme and electrode using pyrene crosslinking chemistry. This study suggests that developed biosensors could be promising for H₂O₂ detection in medical and environmental applications.