The invention belongs to the field of environmental monitoring and particularly relates to a method for determining a subsurface wetland blocking position with plant cellulose oxygen isotope. In an undercurrent constructed wetland zone to be accessed, points are distributed evenly, wholesomely-growing plants are collected, plant cellulose in a subsurface wetland is extracted, a mass spectrograph is used for analyzing the oxygen isotope in the cellulose, and an oxygen isotope abundance isoheight distribution diagram at the subsurface wetland zone to be accessed is drawn. An isoheight dense region in the distribution diagram is the blocking position of subsurface wetland water flow. According to the method, a blocking zone of the undercurrent constructed wetland can be determined quickly and accurately, cost is low, practicability is high, and secondary pollution is avoided.