The invention relates to a method for separating and purifying mussel mucin by using a mixed adsorption chromatography. Mussel mucin contains a group of L-3,4- -Dihydroxyphenylalanine (L-DOPA), a phenohydroxyl group thereof can act as the supplier for hydrogen bond, the benzene ring thereof can generate a hydrophobic effect, and the lysine thereof with strong positive charges is capable of forming a static bond. On the basis of the properties of mussel mucin, a mixed adsorption chromatography (i.e. the adsorption chromatography based on three principles of adsorption with hydrogen bond, adsorption with hydrophobic effect, and static adsorption) is adopted to overcome the problem of low yielding rate of mussel mucin in the prior art for separating and purifying mussel mucin. An strong acid extraction is adopted to eliminate small-molecular compounds from a desalting column, an argar medium with high concentration and high cross-linking degree to separate and purify mussel mucin, and an acetic acid-urea- polyacrylamide gel electrophoresis is used to differentiate mussel mucin through specific chromogenesis with nitro blue tetrazolium. Three principles adopted with one separation medium to separate mussel mucin achieve high selectivity, simplify the purification technology, and decrease production cost.