A new horseradish peroxidase (HRP) third-generation electrochemical biosensor based on ceria nanocubes (CeO(2)-NCs) and chitosan (Chit) was developed. The single-crystalline, uniform and size-controlled CeO(2)-NCs have been synthesized by hydrothermal method. HRP was immobilized in CeO(2)-NCs and Chit film on the glass carbon electrode (HRP/CeO(2)/Chit/GCE). Compared with HRP-chitosan modified electrode (HRP/Chit/GCE), HRP/CeO(2)/Chit/GCE exhibited a pair of more obvious redox peaks at -0.348 V (versus Ag/AgCl). Experimental results indicate CeO(2)-NCs greatly promoted the electron transfer between HRP and GCE. The immobilized HRP exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide (H(2)O(2)). The resulting biosensor showed a linear range of 1-150 microM and a detection limit of 0.26 microM estimated at a signal-to-noise ratio of 3. Stability and reproducibility of the biosensor were also studied. The biological activity of HRP immobilizing in the composite film was characterized by UV-vis and FTIR spectra.