Abstract: ⁷⁹Se, with little radioactivity and little intrusiveness to biological tissue even to cell, is an ideal tracer nuclide for long period and non-intrusive biological research of selenium. Establishment of its tracer methodology is significant in biomedicine and life science. ⁷⁹Se-AMS is an alternative owing to its high sensitivity, accuracy and precision. Tracer experiment using ⁷⁹Se was carried out to investigate the metabolism kinetics of selenium in the rat kidney, and therefore establishment of ⁷⁹Se-AMS biological tracer methodology is detailed, including tracing sample preparation, chemical procedure, and AMS measurement processing. During AMS measurement, molecular negative ions of SeO^-2 were extracted from the ion source, and the counts of ⁷⁹Se were determined from the discrimination between ⁷⁹Se and ⁷⁹Br by four-anode double-grid ionization chamber. Metabolism curve of sub-poisonous selenium in the kidney of Wistar rat was established finally. Further applications of ⁷⁹Se-AMS for biological research will be explored.