A non-enzymatic electrochemical H2O2 sensor was developed by in situ fabrication of biocompatible chitosan (CS) hydrogels, in which a specific recognition molecule for H2O2, thionine (TH), was stably immobilized via one-step electrodeposition. Electron transfer of TH was facilitated in the CS/GPTMS/TH (GPTMS: γ-glycidoxypropyltrimethoxysiloxane) hydrogels with an electron transfer rate constant (ks) of 3.5 ± 0.1 s(-1). Meanwhile, TH in CS hydrogels maintained high electrocatalytic activity toward H2O2. The developed sensor exhibited a fast amperometric response toward H2O2 within 7 s and a linear response for H2O2 ranging from 5.0 × 10(-6) to 6.9 × 10(-4) M with a detection limit of 1.0 × 10(-6) M. In addition, the non-enzymatic electrochemical sensor exhibited long-term stability with a current decrease less than 5.0% in 18 days and good reproducibility with a small deviation of 7.2%. The remarkable analytical performance of the present sensor provided a promising model for durable monitoring of H2O2 in rat brain microdialysates, which is very useful for understanding the biological effects of H2O2 on the pathological and physiological processes.