Abstract: A method for the determination of δ¹³C and δ¹⁵N of urea in yeast fermentation broth by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was established. Urea was derived as 2-methoxypyrimidine by malonaldehydebis (dimethyl acetal) (MDBMA) and diazomethane in acidic condition, and then was analyzed by GC-C-IRMS. Sample pretreatment method and GC conditions were confirmed and optimized by GC-FID. The results show that the linear correlations of labeled level of δ¹³C(0.5% labeled) and δ¹⁵N (0.10% labeled) and measured value of GC-C-IRMS are well. The correlation coefficients of δ¹³C and δ¹⁵N are 0.998 and 0.999, respectively. The repeatability of δ¹³C and δ¹⁵N of urea in yeast fermentation broth are 0.21‰ and 0.30‰, and the reproducibility are 0.23‰ and 0.29‰. δ¹⁵N of urea from three different sources was analyzed by EA-IRMS and GC-C-IRMS, and the correlation coefficient is 1. This method has high accuracy and good repeatability, which can provide foundation and technical support to low stable isotope labeling urea tracer experiment and analysis of urea cycle metabolism.