The invention provides a method for determining the utilization amount of water in various soil layers by trees based on isotope determination. The method comprises the following steps: 1, collecting an isotope sample; 2, determining the isotope sample; 3, determining the water source water utilization rate of trees; 4, determining the stem sap flow; 5, calculating the sap flow flux of a single tree; and 6, determining the water source water utilization amount of the trees. The determination method starts from the water absorption and the water consumption of the trees, the water source and the water source contribution proportion of the trees are determined based on a stable oxyhydrogen isotope technology, and the water consumption of a single tree is calculated through combining a sap flow meter. The method for determining the utilization amount of water in various soil layers by trees allows an accurate determination result to be obtained through calculating an optimum solution by adopting a multivariate mixed linear model (Iso-source) based on reasonable division of the water source, and provides reference for vegetation recovery, tree kind selection, tree kind configuration mode and later maintenance management.