A bioassay-guided fractionation was performed to evaluate the anthelmintic activity of the crude extract fractions and osthole from Radix angelicae pubescentis against Dactylogyrus intermedius in goldfish (Carassius auratus) in vivo. Among four extracts (petroleum ether, ethyl acetate, acetone, and ethanol), only ethanol extract exhibited the best anthelmintic efficacy with 100% mortality of Dactylogyrus and no death of fish at the optimal anthelminthic concentration of 120 mg/L. Therefore, ethanol extract was subjected to column chromatography to obtain sixteen fractions. The activity was found in fraction F with 100% mortality of Dactylogyrus and no toxicity to fish at dose of 2.0 mg/L. A white crystal was isolated from fraction F and identified as osthole which exhibited the optimal activity with 100% mortality of Dactylogyrus at 1.6 mg/L had and no toxicity to fish at dose up to 6.2 mg/L. This is the first report on the isolation and identification of anthelmintic active compound from R. angelicae pubescentis against D. intermedius in goldfish (C. auratus) in vivo.