Abstract: The ¹³C,¹⁵N₂ doubly labeled Urea and ¹⁵N₂ labeled Hydrazine hydrate were used as raw materials, and ¹³C,¹⁵N₃-SEM·HCl was synthesized by a direct one-step synthesis which based on the reflux reacting. The reaction conditions of ¹³C,¹⁵N₃ labeled semicarbazide hydrochloride were mainly investigated by the experiment designs, like singlefactor design and orthogonal design. The following optimized conditions were obtained as follow: the mole ratio ¹⁵N₂-N₂H₄ to ¹³C,¹⁵N₂-urea was 1.4∶1, the reaction temperature was 135 ℃, the reaction time was 4.5 h, and the yield of ¹³C,¹⁵N₃ labeled semicarbazide hydrochloride was above 90%, the purity ≥98%, the abundance of ¹³C ≥97%, the abundance of ¹⁵N ≥99%. This technology had many advantages, like short production cycle, high yield and sample later-processing.