Abstract: As an important radioactive tracer, ¹⁴C has been widely used in many fields, such as biomedicine, agriculture, geology, environment etc, due to its ability to reveal the distribution, metabolic mechanism and migration path of the substances. In recent years, the demand for ¹⁴C-labeled compounds has continued to grow, and its application field has been expanded. This paper summarizes the sources, preparation, separation and purification methods of ¹⁴C isotopes. Meanwhile, this paper reviews the preparation, structural identification and application in related research fields of ¹⁴C labeled compounds, and emphatically discussed the development status and trend of ¹⁴C isotope separation and purification technology. It is hoped that recyclable utilization of ¹⁴C resources can be realized with solving the pollution of ¹⁴C emissions, which will further promote the development of 14C application.