We introduced Ni Apt as the first aptamer with a characterized dissociation constant for recognizing Ni-NTA. Serving as a nucleic acid analog of the His-tag commonly employed for protein purification using Ni-NTA resin, Ni Apt displays a remarkable binding affinity (Kd = 106 nM) towards Ni-NTA. Furthermore, it can be eluted from the resin using imidazole or EDTA, similar to the removal of His-tag from Ni-NTA resin. The versatile capabilities of Ni Apt make it a valuable molecular tool in nucleic acid purification and recognition applications.