Abstract: Objective To screen for the antifungal active substance from water-soluble extract from Asterias amurensis in South China Sea. Methods Freezen-dried Asterias amurensis were crushed, from which the fat soluble substance was removed with organic solvents. The solid components were immersed into distilled water at a low temperature overnight, separated by combined HPLC and sephadex column followed by C18 silicagel column. Activity of anti-fungal substance was detected using micro-dilution method. Results Five fractions were isolated from the water-soluble Asterias amurensis extract by combined HPLC and sephadex column, including first separated fractions 1-5 (FSF-1, FSF-2, FSF-3, FSF-4, FSF-5). Of these first separated fractions, FSF-3 showed a stronger antifungal activity on Tricophyton rubrum whereas FSF-4 revealed a weaker antifungal activity on Tricophyton rubrum, Tricophyton tonsurans and microsporum gypseum. FSF-2, FSF-3, and FSF-5 had no significant inhibitory effect on Tricophyton rubrum, Tricophyton tonsurans and microsporum gypseum. Three additional fractions were separated from FSF-3 by combined HPLC and sephadex column, including secondary separated fractions 1-3 (SSF-1, SSF-2 and SSF-3). The SSF-1 showed no obvious antifungal activity on Tricophyton rubrum whereas SSF-2 and SSF-3 at the dose of 1μg/ml demonstrated significant antifungal activities on Tricophyton rubrum. Conclusion Active water-soluble components against tricophyton rubrum can be separated from Asterias amurensis in South China Sea.