Abstract: Tritium-labeled compounds are widely used in the field of medicine, especially tritium-labeled biomolecules, which can be used to analyze the metabolic processes of various molecules inside and within cells. This article introduces the synthesis methods of tritium-labeled compounds, provides an overview of the achievements and research progress of tritium-labeled biomolecules in the field of radiobiology and medical applications, with a focus on explaining the principles and applications of tritium-labeled thymidine nucleosides in determining DNA synthesis efficiency. Tritium-labeled compounds offer the advantages of simplicity, speed, intuitiveness, and accuracy in studying the distribution and metabolic processes of molecules such as DNA, RNA, and proteins within cells. Further development of analytical methods based on tritium-labeled compounds is of significant importance for a deeper understanding of molecular biology mechanisms.