Hierarchical LiMn2O4 microspheres were prepared using urchin-like α-MnO2 as a self-sacrificial template. As the cathode material for lithium ion batteries, the electrochemical properties were characterized by cyclic voltammetry and charge/discharge experiments. The results indicated that the LiMn2O4 microspheres had high discharge capacity and excellent cycle stability. Furthermore, a new electrochemical biosensor was constructed by immobilization of bovine myoglobin (Mb) and LiMn2O4 in a thin film of chitosan (CTS) on the surface of carbon ionic liquid electrode (CILE). Electrochemical behaviors of Mb entrapped in the film were investigated by cyclic voltammetry. A pair of well-defined redox peaks appears, which indicates a fast electron transfer rate of Mb with the underlying CILE. The electron transfer coefficient and electron transfer rate constant of Mb were calculated to be 0.47 and 1.04s−1, respectively. The Mb modified electrode showed good electrocatalytic ability toward the reduction of trichloroacetic acid (TCA) with a detection limit of 0.16mmolL−1 (3σ).