Abstract: We analyzed the effect of pH-shift processing method on isolation of crustacyanin from white leg shrimp (Litopenaeus vannamei) shell. The recovery rate, purity and secondary structure content of protein were selected as separation characteristics; the protein was recovered by pH-shifting at a 1.0 interval via acid and alkali. Under these conditions, the protein recovery rate reached 47.5% with purity of 78.23%; the molecular weight was 45 000 Da analyzed by SDS-PAGE and Size Exclusion Chromatography; the maximum and minimum solubilities were obtained at pH 3.0 (60.5%), 11.0 (55.7%) and 5.0 (15.4%), respectively; the isoelectric point determined by Isoelectric Focusing Electrophoresis was about 5.6. Secondary structure content obtained by circular dichroism spectroscopy and spectrum revealed that crustacyanin mainly existed in helix form and contained nearly 70.1% helices via pH-shift processing.