Abstract: Steps to isolate strongly parasitic fungi by single-spore was: smear the fungi spores on to the surface of 2% water agar, find a single-spore under microscope, separate the spore by press a capillary (with the diameter of 1mm) into the agar, pick the piece of agar wafer with the spore and then transferred to corresponding medium. This method simplified isolation procedures, improved isolation speed and success rate, and was especially suitable for isolating strongly parasitic fungus. The method was used to isolate Venturia nashicola，Diplocarpon mali and Cercospora circumscissa and more than 60% single-spores developed to colonies.