The quantitative detection of glucose in urine with high sensitivity and specificity is of great significance in clinical diagnosis. This work utilized 4-Cyanophenylboronic acid (4-CPBA) as a secondary carbohydrate probe in a surface-enhanced Raman spectroscopy (SERS) -based urine glucose quantification assay. This assay protocol does not require the conjugation of the probe to the SERS substrate. Furthermore, the characteristic Raman peak of cyano group at 2226 cm-1 was located in the biological silent region (1800-2200 cm-1), thus avoiding interference by other endogenous biological molecules. This method allows high selectivity of glucose over other carbohydrate such as fructose and galactose. This method has been successfully applied for specific detection of urine glucose，with the detection limit down to 10 nM. Furthermore, the glucose content in urine from patients with mild diabetes was successfully detected. The experimental results show that this method offers a highly specific and sensitive way for the determination of glucose in urine，as a potential powerful tool for subsequent quantitative analysis.