Abstract: The majority of soil nitrogen is closely associated with soil organic matter, the cleavage of high molecular weight organic N such as protein to smaller soluble compounds (e.g amino acids) is a key step in the terrestrial N cycle. Therefore, as amino acids occupy a central position in understanding the N biogeochemical cycling processes and ecosystem functions, using natural abundance level isotopic information of amino acids or the heavier isotope 15N as a tracer helps advance the knowledge of soil fertility and its biological mechanism. Currently isotope pool dilution approach and compoundspecific isotope analysis (CSIA) approaches have been successful established to measure amino acid in soils. However, both of them need large sample requirements and laborconsuming isolation procedures for the isotope analysis of amino acids. In this study, a novel distillationchemical conversion N2O approach based on the 15N analysis by the oxidation of amino groups to NO2- and further reduction to N2O was described, followed by PreCon isotope ratio mass spectrometry (PreCon-IRMS). Sample preparation was carried out by our self-developed deviceautomatic micro-nitrogen conversionN2O generating device, allowing either helium (He) purging of gas or liquid charging from a range of suitable, airtight bottles. The reaction conditions for the N2O production were explored and optimized, including the hypobromite concentration, NaOH concentration, and amino acid mixtures concentration. The nitrogen stable isotope abundance in standard solutions of amino acid mixtures with different concentrations and 15N abundance, as well as the nitrogen stable isotope abundance of free amino acids in tested soil were analyzed by using the newly developed pretreatment method combined with the PreConIRMS system. The results showed that the developed method coupled IRMS analytical system was able to measure the 15N abundance in all standard solutions and soil extracts with good precision of 02%08% (CV<1%) without isotope fractionation at the limits of quantification as 0.5 μmol. This method has a great advantage compared with current approaches avoid time-consuming isolation and toxic reagents requirement. The pretreatment analysis time is short, and the soil samples with the amino acid concentration of nmol grade can be detected.