Subcritical water extraction (SWE) of fucoidan from Saccharina japonica was investigated. To optimize, a Box-Behnken design (BBD) was used to study the influences of temperature [100–180 °C], pressure [20–80 bar], solid to liquid (S/L) ratio [0.04–0.09 g mL−1], agitation speed (AS) [100–300 rpm], and reaction time (RT) [5–15 min]. By unraveling the regression equations, the optimal conditions were 127.01 °C, 80 bar, S/L ratio of 0.04 g mL−1, AS of 300 rpm, and RT of 11.98 min. Under these conditions, the crude fucoidan (CF) yield was 13.56%. FT-IR, monosaccharides, TGA and UV–Vis confirmed that the extracted polysaccharides had features of fucoidan. CF showed good antioxidant activities, while anti-proliferative activity showed a moderate cell proliferation for few cell lines. In conclusion, a high yield of CF with good functional activity obtained from SWE might be the favored method for industrial scale production of fucoidan.