In this paper, a new probe is proposed for the in vivo dielectric measurement of anisotropic tissue in radio frequency band, which could accomplish the dielectric measurement in perpendicular directions by one operation. The simulative studies are performed in the frequency range from 1–1 000 MHz in order to investigate the influence of probe dimension on the energy coupling and sensitivity of measurement. The suitable probe is designed and validated for the actual measurement in this frequency band. According to the simulation results, the energy coupling of the probe could be kept below –12 dB in the frequency range from 200–400 MHz with high sensitivity of measurement for the dielectric properties of anisotropic tissue. That indicates the new type of probe has the potential to achieve the dielectric measurement of anisotropic tissue in radio frequency band and could avoid the measurement error by multi-operations in the conventional method. This new type of probe could provide a new method for the in vivo dielectric measurement of anisotropic tissue in radio frequency band.