We identified an efficient protocol for extracting proteins from whole earthworm, Eisenia fetida, for 2-DE. Sample preparation is a critical step in a 2-DE proteome approach and is absolutely essential for obtaining good results. Six protein extraction protocols based on different protein precipitation agents were tested and evaluated using 2-DE. The methods generated remarkably different 2-DE protein spot patterns. We conclude that trichloroacetic acid (TCA)-A eliminates interfering compounds, thus allowing for the efficient resolubilization of proteins. TCA-A gives good distinction, more bands in 1-DE gels, and the most number of protein spots in 2-DE gels. It is also rapid, provides the higher protein yield, and has the less number of steps. To demonstrate the quality of the extracted proteins, we cut several protein spots that were common to four methods from 2-DE gels, analyzed them using MALDI-TOF/TOF MS, and tentatively identified them. The classic TCA-A method proved to be most useful as a standard method of extracting proteins from E. fetida.