This study investigated the potential of dual-ligand hydrophobic charge-induction chromatography for the simultaneous recovery of two high-value components (bovine serum albumin and bovine immunoglobulin G). Compared to single-ligand chromatography, dual-ligand chromatography offers greater flexibility and room for improvement. Tryptophan and tryptamine were used as ligands to prepare a series of single- and dual-ligand resins. Bovine serum albumin and bovine immunoglobulin G were used for adsorption and comparison in batch, frontal adsorption, and recovery experiments. The results showed that the dual-ligand mode had a stable coupling efficiency ratio (tryptamine to tryptophan, ∼2.30) during preparation and could break through the upper limit of the adsorption capacity of single-ligand mode. Additionally, it exhibited good adsorption performance for both bovine serum albumin and bovine immunoglobulin G from bovine serum (recoveries of 90.73 and 101.4%, respectively). The findings of this study confirmed that dual-ligand chromatography can be used as an effective strategy for biological separation.