A new approach for estimation of the tree root biomass is proposed by using GPR system with high accuracy positioning system. The 3D images of subsurface can be obtained clearly with this system, which we refer as "3DGPR". We try to estimate the biomass of tree roots quantitatively by measuring the volume of the tree roots. We tested this system for tree roots measurement, and the broadening of the tree root with horizontal direction could be detected with 500 MHz and 800 MHz antenna. We excavated this tree every 10 cm from 0 cm to 50 cm to validate the accuracy of the result. Compared with the measurement result and the excavated one, the tree root whose diameter is more than 5 cm could be detected correctly. Some tree roots whose diameter is 3 cm also could be detected. The diameter in most of the tree roots is more than 2cm. In this result, we expect that the volume is supposed to be estimated within 30 percent error by using the excavated result for calibration data.