Abstract: Mitochondria are the major sites of respiration and energy metabolism in plants. However, previous methods for measuring respiration metabolism of mitochondria are complex and time- consuming. Thus, accurate and efficient methods are in urgent need for measurements of mitochondrial respiration. In the current study, we developed a simple and efficient method to test respiration metabolism of mitochondria in living cells and tissues by using Extracellular Flux Analyzer. Oxygen consumption rates were identified quickly in floral cells of Magnolia denudata, which is a perennial woody plant. In the measurements, stable repeated data of samples were obtained, indicating the reliability of this method in measuring mitochondrial respiration and energy metabolism. Furthermore, respiratory pathways were identified in the cells via using various respiratory inhibitors. In addition, the method allows dynamic and real-time monitoring of mitochondrial respiration in plant tissues and individual plant cells, which outperforms existing methods. Above all, this method shows a broad application potential in measuring mitochondrial energy metabolism of plant cells in the future.