In this study, a new matrix for immobilized metal affinity chromatography (IMAC) using poly(2-acetamidoacrylic acid) (PAAA) hydrogels complexed with Ni 2+ was developed for the purification of the recombinant histidine-tagged green fluorescence protein (His6–GFP). The Ni 2+ -complexed PAAA hydrogel was prepared by polymerizing 2-acetamidoacrylic acid (AAA) and 2,2′-[(1,4-dioxo-1,4-butanediyl)diamino] bis(2-propenoic acid) (DBDBPA) with potassium persulfate in DMSO, followed by Ni 2+ complexation. Confocal laser scanning microscopy was used to determine the binding of His6–GFP to the Ni 2+ –PAAA hydrogel in three-dimensional space. Photoluminescence spectroscopy revealed an 81% binding efficiency of His6–GFP to the Ni 2+ –PAAA hydrogel yielded with a recovery of 59%. The specificity of His6–GFP binding to Ni 2+ –PAAA hydrogel was compared with that of the PAAA hydrogel without Ni 2+ . His6–GFP was purified directly from the cell lysate with Ni 2+ –PAAA hydrogel matrix but the PAAA hydrogel without Ni 2+ had no effect. The major advantage of the Ni 2+ –PAAA hydrogel system over current methods, such as Ni–nitrilotriacetic acid (NTA) agarose beads, was the simple and low-cost procedure for preparing the matrix.