Abstract: A rapid method was established for the determination of isotope abundance of ¹³C labeled urea by enzymatic method. Two-step reaction was carried out in a specially made reaction tube with three bulbs. In the first step, urea-¹³C was catalyzed to form ¹³C labeled ammonium carbonate at 40℃ for 5 min. In the second step, ¹³C labeled carbon dioxide was formed by acidification. The gas was introduced into MAT-271 gas isotope mass spectrometer to detect the ion current intensity with mass-to-charge ratio of 44 and 45, and the ¹³C isotope abundance was calculated. The results showed that the error between the measured value and the labeled value of the standard sample was within 0.1% ¹³C, and the precision RSD was less than 0.1%. The results of the established determination method were basically consistent with those of the current standardization method. The method was relatively simple, rapid and specific, which was suitable for rapid determination of isotope abundance of ¹³C labeled urea in breath test diagnostic reagents and active pharmaceutical ingredients.