Abstract: Carbon plays an important role in the metabolism of plants and human. Radiocarbon (¹⁴C) is an ideal tracer for vivo human metabolism, disease diagnosis and organ function assessment studies. In this paper, the preparation method of ¹⁴C breath test samples for accelerator mass spectrometry (AMS) measurement was developed in China. AMS, with its advantages of high sensitivity, small radioactivity dose, high precision, large measuring range, and long tracer cycle, can be used to measure cosmogenic nuclide ¹⁴C, which has long half-life. The current intensity of interested ion in ion source is an important parameter for the sensitivity of AMS measurement. The high beam current can improve the sensitivity of AMS. The preparation methods of breath test samples of ¹⁴C tracer were systematically studied to obtain high beam current using graphitization and carbonatization methods. A reliable preparation procedure of breath test samples for ¹⁴C tracer and its optimization parameters were determined by testing beam currents of various samples, which has laid a foundation for ¹⁴C-AMS technology at breath test tracer application.