The new ion-imprinted guanyl-modified cellulose (II.Gu-MC) was prepared for the separation and determination of Cu (II) ions in different real samples. Several techniques such as Fourier Transform Infrared (FT-IR), scanning electron microscope (SEM), thermal analysis, potentiograph and elemental analysis have been utilized for the characterization of II.Gu-MC. The adsorption behavior of the ion imprinted polymer (II.Gu-MC) was evaluated and compared to the non ion-imprinted polymer (NII.Gu-MC) at the optimum conditions. The selectivity and the adsorption capacity were greatly enhanced by using the ion-imprinted polymer, indicating its validation for the separation and determination of Cu²⁺ ions in different matrices. The adsorption capacity by chelating fibers II.Gu-MC & NII.Gu-MC agreed with the second-order model, and the sorption-isotherm experiments revealed best agreement with Langmuir model. The adsorption capacity of II.Gu-MC and NII.Gu-MC were 115 and 55 mg·g⁻¹, respectively. The II.Gu-MC was successfully employed for the selective separation and determination of Cu(II) ions with high accuracy.