The multifunctional bioactive materials were prepared from Enteromorpha prolifera by enzyme-assisted extraction using four proteases and seven carbohydrases, and the biological activities of the enzyme-assisted extracts were evaluated as antioxidant, anti-acetylcholinesterase (AChE) and anti-inflammatory effect as the measures of inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells. The enzyme-assisted extracts were rich in polyphenols in the range 124 ± 4.2 to 844 ± 9.1 mg/100 g and flavonoids in the range 453 ± 6.0 to 675 ± 5.2 mg/100 g, and Protamex and Viscozyme extracts, which were rich in polyphenols and flavonoids, showed the highest 2,2-diphenyl-1-picrylhydrazyl scavenging, hydrogen peroxide scavenging, ferrous ion chelating and reducing power. Flavourzyme extract (89.92%) and Promozyme extract (93.64%) showed the highest AChE inhibitory activities at the concentration of 1.0 mg/ml. All enzyme-assisted extracts showed no cytotoxic effect on RAW264.7 cells at the tested concentration and significantly inhibited the LPS-induced NO production in RAW264.7 cells.