The use of 1,6-diaminohexane, 1,10-diaminodecane, 1,6-diaminohexane + ethylene glycol diglycidyl ether (EGDGE), and 1,10-diaminodecane + EGDGE as spacer arms for the immobilization of Cibacron Blue 3GA onto Immobilon AV membranes was investigated in this work. The dye ligand density was optimized by the employment of 1,6-diaminohexane, followed by 1,10-diaminodecane, 1,10-diaminodecane + EGDGE, and 1,6-diaminohexane + EGDGE. The adsorption properties of lysozyme onto the dye-affinity membranes were also studied. The results show that the use of 1,10-diaminodecane as a spacer molecule appears to offer the best adsorption capacity and hence results in the highest percentage of ligand utilization. Consequently, a 12-atom straight molecule such as 1,10-diaminodecane is a good choice for dye-affinity membrane separations.