In this work, a novel enzyme-mimicking nanocomposite of Mn(II)-poly-L-histidine (PLH) functionalized carboxylated multi walled carbon nanotubes (CMWCNTs) was designed and synthesized. Based on the catalase-like activity of the nanocomposite, a non-enzymatic hydrogen peroxide (H2O2) biosensor was then established and explored for H2O2 electrochemical detection. The nanocomposite was characterized by Fourier transform infrared spectra, Raman spectroscopy, and transmission electron microscopy. Due to the enlarged effective surface area and the efficient electrocatalytic activity of the Mn(II)-PLH redox-active units, the obtained Mn(II)-PLH-CMWCNT electrode showed excellent electrocatalytic performance toward H2O2 disproportionation. Under the selected optimum conditions, the prepared biosensor exhibited highly sensitive response toward H2O2, and the response current had a good linear relationship between the response currents and H2O2 concentrations in the range of 0.002–1.0 mM, a low detection limit of 0.5 μM and a sensitivity of 464.18 μA mM-1 cm-2. With the good stability, reproducibility and selectivity, the proposed biosensor was successfully applied to the determination of H2O2 in real-life samples, and showed satisfactory results. In summary, the Mn(II)-PLH-CMWCNT nanocomposite could be a promising enzyme-mimicking nanomaterial for the researches of electrocatalysis, biosensing and relevant fields.