By using different spacer arms to immobilize Cibacron Blue 3GA onto regenerated cellulose membrane discs, this work investigated their effects on ligand density and corresponding lysozyme adsorption. When EGDGE alone was adopted as a spacer arm, using 0.1 M EGDGE without the formation of liquid film in its reaction with the membrane disc led to higher adsorption capacities. When EGDGE was combined with another reagent with two end functional groups as a spacer arm, EGDGE + 1,4-diaminobutane performed better in both ligand density and adsorption capacity. In addition, the ligand densities using the combinations of EGDGE with another reagent were generally lower than those from EGDGE alone, whereas the Ligand utilization percentages were higher. Moreover, adding a spacer arm did not affect the binding strength between lysozyme and Cibacron Blue 3GA, regardless of the type of spacer arms and their length, and the reaction condition.