A novel difference value method combining glucose phosphorylation with surface-enhanced Raman scattering (SERS) for ATP quantification is proposed. The quantitative determination of glucose is achieved based on SERS rather than the traditional spectrophotometry. The proper concentration of glucose for ATP quantification is demonstrated to be 1–1000 times of that of ATP. The accuracy of the difference value method is verified, and the limit of detection of ATP reaches as low as 4.69 × 10−7 M.