A method to realize surface-enhanced Raman spectroscopy (SERS) at a titanium alloy substrate for glucose detection has been experimentally demonstrated. A silver-coated laser-induced periodic surface structure (LIPSS) was prepared via femtosecond laser micro-processing. The low detection limit of glucose is 10-7mol/L and, a good linear relationship between the glucose concentration and Raman intensity is found in the range between 1×10-7 and 1×10-3mol/L. Moreover, we investigate SERS detection for glucose sensing in human urine samples, while the results are in good agreement with clinical results. The Letter provides a facile method for producing a structure-controlled SERS substrate to realize glucose detection, which is promising for long-term in vivo diagnostics.