A new procedure for detection of tree roots and root biomass estimation by using a promising Ground Penetrating Radar (GPR) system with high accuracy positioning system was proposed. This system acquires the accurate antenna position and fuses it with the GPR data, and then a three dimensional (3D) data cube can be obtained. After analysing the acquired 3D cube in field measurement, some specific reflection characteristics of roots were summarized, and then a detection procedure was proposed to detect the root extension starting from the tree stump. This detection procedure could exclude the influence of the strong clutters and overcome the weak reflection from one part of the root which resulted from the error of antenna operation in the survey. In order to validate the performance of detection and estimation, one tree was excavated after the measurement. The detection result shows that the coarse roots whose diameters were more than 5cm could be detected correctly, and the extension of the detected roots was corresponding to the true scene. The diameter of roots was over estimated, and the detection procedure needs to be improved in order to reduce the biomass estimation error within 30 percent.