Engraulis japonicus cooking liquid is a by-product from fish processing. It contains various components and rich of nutrients. In order to purify the cooking liquid, chitosan was used as a safe flocculant to separate and recycle proteins from cooking liquid and help membrane concentrating efficiency in the later process. The effect of Chitosan on the flocculation of water soluble protein in cooking liquor was studied under different pH conditions. The analysis of solubility curve, infrared spectrum, molecular weight distribution, heavy metal and amino acid content showed that: the flocculation effect was the best at pH=3, and the removal rate of water soluble protein was 36.23 %. However, considering of the simplicity and economics in the industry, pH=5.5 was selected to move about about 30% water soluble protein. The flocculation mechanism of chitosan was different in different pH environment. In acidic environment, it was mainly depended on charge neutralization and bridging effects to form crosslinking structure between protein and chitosan; under alkaline conditions, the adsorption was dominant in the flocculating process. The pH affected the flocculation of macromolecular water-soluble proteins with chitosan, while the water-soluble proteins whose molecular weight was less than 19kDa remained unaffected. In addition, chitosan flocculation would reduce the content of heavy metals, but it does not affect the change of amino acid content.