Four kinds of colophony were used to isolate the herbicidal substance from the toxin produced by isolate PAM1 of Pythium aphanidermatum, which had herbicidal activity to Digtaria sanguinealis L and the results showed X-5 was the best one for absorbing herbicidal substance among the four kinds of colophony used. Four solvents including 50% ethanol, 95% ethanol, ethyl acetate and acetone were used as eluting solvent and 2 procedures were tested, and the results of growth inhibition and seed germination indicated that the best eluting procedure was procedure 1.