Dynamic and accurate monitoring of cell-released electroactive signaling biomolecules through electrochemical techniques has drawn significant research interest for clinical applications. Herein, the functionalized carbon nanotubes (f-CNTs) featuring with gradient surface wettability from hydrophobicity to hydrophilicity, and even to superhydrophilicity, were regulated by thermolysis of an ionic liquid for exploration of the dependence of surface wettability on electrochemical biosensing performance to a cell secretion model of hydrogen peroxide (H2O2). The superhydrophilic f-CNTs demonstrated boosting electrocatalytic reduction activity for H2O2. Additionally, the molecular dynamic (MD) simulations confirmed the more cumulative number density distribution of H2O2 molecules closer to the superhydrophilic surface (0.20 vs 0.37 nm), which would provide a faster diffusional channel compared with the hydrophobic surface. Thereafter, a superhydrophilic biosensing platform with a lower detectable limit reduced by 200 times (0.5 vs 100 μM) and a higher sensitivity over 56 times (0.112 vs 0.002 μA μM cm-2) than that of the hydrophobic one was achieved. Given its excellent cytocompatibility, the superhydrophilic f-CNTs was successfully applied to determine H2O2 released from HeLa cells which were maintained alive after a 30 min real-time monitoring test. The surface hydrophilicity regulation of electrode materials presents a facile approach for real-time monitoring of H2O2 released from living cells and would provide new insights for other electroactive signaling targets at the cellular level.