The objectives of this study were to detect coarse tree root and to estimate root biomass in the field by using an advanced 3D Ground Penetrating Radar (3D GPR) system. This study obtained full-resolution 3D imaging results of tree root system using 500 MHz and 800 MHz bow-tie antennas, respectively. The measurement site included two larch trees, and one of them was excavated after GPR measurements. In this paper, a searching algorithm, based on the continuity of pixel intensity along the root in 3D space, is proposed, and two coarse roots whose diameters are more than 5 cm were detected and delineated correctly. Based on the detection results and the measured root biomass, a linear regression model is proposed to estimate the total root biomass in different depth ranges, and the total error was less than 10%. Additionally, based on the detected root samples, a new index named “magnitude width” is proposed to estimate the root diameter that has good correlation with root diameter compared with other common GPR indexes. This index also provides direct measurement of the root diameter with 13%–16% error, providing reasonable and practical root diameter estimation especially in the field.