Extraction of proteins from pine needles for proteomic analysis has long been a challenge for scientists. We compared three different protein extraction methods including sucrose, Tris-HCl and trichloroacetic acid (TCA)/acetone (TCA method) to determine their efficiency in separating pine needle proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional PAGE (2D-PAGE). Proteins were then separated by SDS-PAGE. Among three methods the method using sucrose extraction buffer showed the highest efficiency and highest quality in separating proteins. In addition, clearer and more stable strips were detected by SDS-PAGE using sucrose extraction buffer. When the proteins extracted using sucrose extraction buffer were separated by 2D-PAGE, more than 300 protein spots, with isoelectric points (PI) ranging from 4.0 to 7.0 and molecular weights (MW) from 6.5 to 97.4 kD, were observed. This confirmed that the method with sucrose extraction buffer was an efficient and reliable method for extracting proteins from pine needles.