Raman spectroscopy offers a rapid and nondestructive method for qualitatively and quantitatively analyzing the molecular structure of substances. Herein, a facile and cost‐effective approach for the preparation of three‐dimensional (3D) surface‐enhanced Raman scattering (SERS) substrates is proposed, in which pyramid structures are fabricated using silicon anisotropic wet etching and silver dendrites are decorated on these silicon pyramid (PSi) substrates by immersing them in a mixture of hydrofluoric acid and silver nitrate for several minutes. The 3D SERS substrates, based on PSi coated with Ag dendrites, reveal high‐performance SERS enhancement (with an analytical enhancement factor reaching 3.54 × 1010) and can detect rhodamine 6G (R6G) in the presence of chemical enhancer (lithium chloride) at low concentrations down to 10−11 m. For glucose detection, 2‐thienylboronic acid is employed to faciliate glucose attachment to the substrate surface, achieving a detection limit as low as 10−6 m. The substrate exhibits good repeatability with a relative standard deviation of 11.223%, as well as reusability and long‐term stability for detection. The results also highlight the excellent sensitivity of the PSi‐based SERS substrate, which is expected to be instrumental in biochemical analysis and holds significant potential for developing noninvasive glucose sensor for diabetic patients using saliva samples. The three‐dimensional silver dendrites/silicon pyramid surface‐enhanced Raman scattering (SERS) substrates are successfully fabricated by anisotropic wet etching of a silicon wafer and chemical deposition in a mixed solution (HF/AgNO3). The proposed SERS substrates can detect glucose at ultralow concentrations below 10−6 m and exhibits high repeatability as well as reusability and long‐term stability for detection.