Heparan sulfate (HS), one kind of sulfated glycosaminoglycans (GAGs), plays an important role in growth factor-receptor interaction, thereby involving in growth, angiogenesis and innate immune signaling. It is also highly addressed that GAGs and their anchoring core proteins are abnormally expressed in various cancers including lung, liver, and colon cancers. Human ribonuclease A (RNaseA) family members, hRNase2, hRNase3 and hRNase5 are reported to bind to cell surface by recognition of sulfated GAGs. We have recently identified three functionally important HS and heparin binding regions in hRNase3, also named as human eosinophil cationic protein (ECP), and characterized a dual-functional peptide (CPPecp) with GAG-binding and cell-penetrating activities. Screening of in vitro binding activity on various gastrointestinal cell lines demonstrated that CPPecp bound to cells rich in HS on the cell surface. However, cellular binding activity of FITC-CPPecp was abolished while cell surface HS was removed by haparinase treatment. In order to characterize the specific binding of CPPecp, quantitative assay was carried out to precisely decipher binding affinity between CPPecp and a variety of GAGs. In addition, magnetic nanoparticle (Fe3O4)-conjugated CPPecp (MNP-CPPecp) was synthesized as a novel bioprobe to evaluate its feasibility in in vivo magnetic resonance imaging (MRI) analysis in colon tumor mouse model. Taken together, our CPPecp in vitro preferred to bind to sulfated GAGs and selectively attach to HS on cell surface. Moreover, CPPecp demonstrated in vivo tumor targeting activity in colon tumor mouse model, suggesting that CPPecp possesses high potential to be developed as a novel molecular imaging agent and cancer targeting delivery vector.