The direct absorption and utilization of low-molecular weight organic nitrogen (N) by soil microbial is a new subject in the research of microbial N nutrition. The study used gas chromatography-mass spectrometry to trace dual-labeled (13C, 15N) glycine from the soil solution and microorganisms. The results showed that glycine added to the soil was quickly taken up by soil microorganisms, with the half-life of glycine being 2.9 h. Withthe incubation of 4 h, the maximum amount of dual-labeled glycine in the microbial biomass was measured (equivalent to 10% of glycine added), indicating that added glycine was absorbed as intact molecular by soil microorganisms. The single labeled-Keto acid was detected in soil solution and in the microorganisms (decomposed production by double labeled glycine), but the content is extremely low, suggesting that added glycine mainly served as carbon (C) source for soil microbial life activities. This study demonstrated that compound specific stable dual labeled isotope analysis combined with chloroform fumigation technique was an effective method for detecting the low-molecular organic N utilized by soil microorganisms.