Girl Talk doesn’t actually isolate sounds; it’s almost impossible to do that. You can use EQ to quiet background sounds to a limited extent, but there’s no way to perfectly isolate sounds from a mixed track. Girl Talk and other mashup artists rely heavily on a capellas (vocal-only mixes) and instrumentals, which are included with DJ versions of most pop, hip-hop and dance tracks. A few isolated stems have also been leaked from the Rock Band and Guitar Hero games, they pop up online here and there. Every so often an artist will release tracks separated into stems officially. Kanye West even gives away stems of “Love Lockdown” for free on his blog.
Girl Talk uses comparatively primitive software for his mashups, so his process is labor-intensive. If you want to make life easier for yourself, get a hold of Ableton Live. Once you get a hang of the user interface, it’s a wonderfully versatile sampling and remixing tool. Before I got Ableton, I used a combination of various audio editors, Recycle and Reason, which worked well enough, but was a pain. Ableton is better.