The invention relates to a preparation method of an electrochemical glucose biosensor and a detection method for testing glucose. The preparation method includes the steps of dispersing calcium titanate nanometer particles in a chitosan solution to prepare a GOD/CaTiO3/CS mixed solution; modifying the surface of a preprocessed glassy carbon electrode with the prepared GOD/CaTiO3/CS mixed solution to obtain the electrochemical glucose biosensor. The method for detecting a glucose sample through the electrochemical glucose biosensor prepared through the preparation method includes the following steps of firstly, adding the glucose sample to test liquid with the prepared modified electrode as the working electrode, a saturated calomel electrode as the reference electrode and a platinum sheet electrode as the counter electrode; secondly, detecting electrochemical signals of the working electrode through an electrochemical working station. Glucose oxidase is fixed through calcium titanate nanometer particles, and the biology catalytic activity of biological macromolecules can be well kept.