Abstract: Three fructo-oligosaccharides (FOS), including fructo-oligosaccharide DP7, DP8 and DP9, were determined by ¹H nuclear magnetic resonance spectroscopy (¹H-NMR), using sucrose as the internal standard substance and deuterium oxide as the solvent. The optimized ¹H-NMR parameters were as follows: temperature at 25 ℃, spectral width of (2-14)×10^-6, pulse angle at 45°, pulse delay time of 5 s, sampling time of 3 s, and scanning 16 times. Tests for instrument precision and repeatability of fructo-oligosaccharide DP7, DP8 and DP9 samples were made, with RSDs (n=6) of the determined values less than 0.50%. The contents of three samples were determined by this method, and the results were compared with those obtained by the mass balance method. After evaluation by the F-test, there was a significant difference between the two methods. However, as quantitative nuclear magnetic resonance spectroscopy was usually used for determining absolute content, it could directly determine the components to be tested with only common chemicals, which could support the assignment results of the mass balance method.