A novel single-layer polycrystalline anatase TiO2 (SLP TiO2) nanosheet with porous structure was synthesized through a simple solvothermal method by employing rod-like titanyl sulfate as the starting material in the presence of glycerol, followed by calcination. SEM, TEM, X-ray diffraction, FTIR spectroscopy, thermogravimetry coupled to FTIR spectroscopy, solid-state C-13 NMR spectroscopy, and N-2 adsorption/desorption processes were employed to investigate the structure of the products. The structure and morphology were found to be dependent on the experimental conditions, such as solvothermal reaction time, morphology of titanyl sulfate, and solvent type. The obtained SLP TiO2 nanosheet was employed as a support for loading well-dispersed Ag nanoparticles and proved to be a good surface-enhanced Raman scattering (SERS) substrate for the detection of 4-aminothiophenol and Rhodamine 6G, showing great potential as a convenient and powerful substrate for SERS studies in the field of detecting different target molecules due to good uniformity and reliability.