161Tb is a promising radionuclide owning to its favorable properties for treatment small size of cancer. No Carrier Added (NCA) 161Tb can be produced by indirect method through 160Gd(n,γ) 161Gd→161Tb nuclear reaction. To obtain the NCA radionuclides, the existence of an effective Gd/Tb separation method is critical. In this study isolation of Tb from Gd/Tb matrix using Ln resin column based on extraction chromatography method has been carried out. Fractions eluted from the column containing Gd/Tb matrix were identified and quantified using ICP. The optimization of different experimental parameters for the effective separation of Gd/Tb such as concentration of eluting solotions and flow rate of load and elution was investigated. The results showed that optimum condition on Gd/Tb isolation was obtained using HNO3 solution with concentration of 0.8 and 3 N to separate gadolinium and terbium isotope, respectively. The separation yield of Tb and Gd were obtained 83.51 % and 81.8% respectively.