A novel, biocompatible, thermally steady, and nontoxic zirconia enhanced grafted collagen tri-helix scaffold was prepared on a graphite electrode. This scaffold provided a microenvironment for loading biomolecules and helped to retain their natural structure. UV-vis spectroscopy and scanning electron microscopy were used to characterize the scaffold and the structure of immobilized biomolecules. Using horseradish peroxidase (HRP) as an example, this scaffold accelerated its electron transfer and led to its direct electrochemical behavior with a good thermal stability up to 80 C. The surface electron-transfer rate constant of the immobilized HRP was (5.55 +/- 0.43) s(-1) in 0.1 M pH 7.0 PBS at 18 degrees C. The immobilized HRP showed an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without aid of an electron mediator. The linear response range of the biosensor for H2O2 was from 1.0 to 73.0 mu M with a correlation coefficient of 0.999 (n = 14), a limit of detection down to 0.25 mu M and an apparent Michaelis-Menten constant of (0.28 +/- 0.02) mM. The biosensor exhibited high sensitivity, acceptable stability, and reproducibility. The ZrO2 grafted collagen provided an excellent matrix for protein immobilization and biosensor preparation.