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 carbon screen-printed electrodes (CSPEs) 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 screen-printed electrodes (SPEs). The detection of H2O2 was achieved with HRP/cut-SWCNT/CSPE electrode configuration using voltammetry technique. The constructed biosensor has sensitivity and apparent Michaelis-Menten constant (K-m) values of 34.1 mu A L mmol(-1) cm(-2) and 0.47 mmol L-1, respectively within the linear range of 20 to 600 mu mol 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. (c) 2019 Society of Chemical Industry