How to quantify the root water uptake from different soil layers under alternate furrow irrigation (AFI) condition accurately, is still of great challenge. In this study, the stable hydrogen and oxygen (δD and δ18O) isotopes of soil water, stem water, rainfall and irrigation water (groundwater) in the maize field system under AFI condition were measured during 2013–2014. The root water uptake was estimated by the multi-source mass balance method (IsoSource model). Results showed that δ18O distribution varied significantly between the ridge (R) and furrow (F) near soil surface, and between ridge surface and wet furrow under AFI. δ18O in stem water had better response to soil water variation at wet side, and soil water in wet side was firstly absorbed by maize root and then transported to the dry side. Maize water uptake was mainly from the depths of 20–40 cm at the late jointing stage, 40–60 cm at the late heading stage and 40–80 cm at the filling stage, with relative contribution of 84, 80 and 66%, respectively. Maize water uptake from different soil zones varied remarkably at AFI. For example, at the filling stage, 16–52% of maize water uptake was from the depth of 60–80 cm below the ridge (dry side) before irrigation, 32–78 and 14–64% from the surface soil (0–20 cm) of wet furrow and the ridge at 1 day after irrigation, 14–60 and 8–54% from the depths of 20–40 cm below the ridge side and 40–60 cm below wet furrow at 3 day after irrigation, and 6–80 and 30–60% from the depths of 60–80 cm below the ridge and 40–60 cm below the ridge side at 7 day after irrigation. Thus maize root water uptake was from deeper soil with the advance of growth stage, and it was mainly from the deeper soil in dry side and the shallow soil in wet side under AFI.