Recognition and determination of ubiquitin (Ubq) is very important since it affects the enzymatic mechanisms, which causes damages in the living organisms. This can lead to diseases especially in neuronal system. Molecularly imprinted polymers (MIPs) are selective resins that exhibit high affinity toward target compounds. MIPs have attracted significicant research interest because of their various applications in solid phase extraction, biosensors and drug delivery, etc. Various MIP types can be used such as thermo-sensitive, pH-sensitive, biomolecule-sensitive, ion-sensitive and photo-sensitive in these applications. Photosensitive MIPs can be used to increase the selectivity and binding efficiency to decrease denaturation of target biomolecules. The aim of this study is to prepare photosensitive cross-linked p(Ubq-co-MACys-Ru(bipyr)(2)-MACys) polymeric nanoparticles (UbqINPs) and leach with 0.5 M HCl to create Ubq memories onto nanoparticles (UbqINPs) and optimize their binding parameters in cryogel based column system. For this purpose, different factors such as pH, flow rate, ionic strength and temperature were investigated, and maximum ubiquitin binding capacity was found to be 25 mg/g at pH 7. The results showed that the UbqINPs can be considered as a good binding material for recognition and separation of Ubq.