We developed a highly sensitive non-enzymatic biosensor based on hybrid Ag-Cu2O@TNTs nanostructure aggregate for the detection of cholesterol. A facile anodization process on a thin titanium alloy (Ti6Al4V) plate was achieved to deposit TiO2 nanotubes (TNTs). Subsequently, TNTs was decorated with Cu2O nanomaterials (NM) using wet chemical bath deposition (CBD) technique. The electro-deposition of silver (Ag) nanoparticles on Cu2O@TNTs hybrid aggregate produced high electroactive surface area which enhanced the electron transfer rate. The structure of Ag-Cu2O@TNTs was examined using X-ray diffraction (XRD), transmission emission microscopy (TEM), and field emission scanning electron microscopy (FE-SEM). Cyclic voltammetry (CV) and amperometry techniques were utilized to study the electrochemical behaviour of nanostructures. The hybrid nanostructure demonstrated great sensitivity (12140.06 μAmM−1cm−2) compared to pure TNTs and Cu2O@TNTs with low detection limit (0.057 mM) and fast response. This study highlights exciting potential of nanocomposites in advancement of reproducible and selective biosensor applications.