The exploitation of multifunctional nanomaterials used for colorimetric biosensing assays is highly desired in diagnostic applications. In this paper, a novel TiO2-encapsulated Au (Au@TiO2) yolk-shell nanostructure was synthesized via a simple and controllable method, in which the TiO2 nanotubes could effectively restrain the aggregation and growth of Au nanoparticles and supply more active sites and high surface areas for biocatalytic reactions. The microstructure and composition of as-prepared nanocomposites were investigated by X-ray diffraction (XRD), transmission electron microscopy (TEM), high resolution TEM (HRTEM), and X-ray photoelectron spectroscopy (XPS). The results demonstrated that unique Au@TiO2 yolk-shell nanostructure with the well-dispersed Au nanoparticles confined in nanotube was obtained. The investigation of peroxidase-like activity of Au@TiO2 nanocomposites confirmed that the relative catalytic kinetic followed Michaelis–Menten behaviors. On the basis of the highly efficient peroxidase-like activity of Au@TiO2, an effective method for colorimetric detection of H2O2 and glucose was established. Considering numerous advantages including reduced aggregation, good chemical stability and high catalytic activity available by the present Au@TiO2 with unique structure, designing other yolk-shell nanostructures with exact function orientation using a similar strategy may also be very promising in the application of biocatalysis and bioassays.