Immunoglobulin of egg yolk (IgY) was separated from fresh hen eggs by three steps, water dilution, salt-out precipitation, and hydrophobic charge induction chromatography (HCIC). In the purification step, HCIC was used as a novel technology and two chromatographic operation modes, packed bed and expanded bed, were compared to improve the separation efficiency. For packed bed mode, IgY were separated with the purity of 92% and the recovery of 58.4% at the operation condition of pH 7.5 and sample (total protein concentration of 11.6 mg/mL, IgY purity of 31%) loading of 0.2 mL/mL adsorbent. For expanded bed mode, a similar purity of 90% and the recovery of 59.5% were found at the fluid velocity of 500 cm/h and same sample loading of 0.24 mL/mL adsorbent at pH 7.5. The results demonstrated that HCIC in expanded bed mode is a potential platform technology for antibody separation both in high efficiency and low cost.