Objective To evaluate the efficiency of protein preparation for human esophageal tissue protein diluted in PBS by ultracentrifugation and 7 protocols for protein precipitation. Methods Eight protocols, which included ultracentrifugation and 7 protocols for protein precipitation ［ammonium sulfate, chloroform/methanol, acidified acetone/methanol, ethanol, acetone, trichloroacetic acid (TCA), TCA/acetone］, were used to extract PBS-diluted mixed protein samples derived from human esophageal cancer tissue (10 cases). Protein concentration was determined by bicinchoninic acid(BCA), Bradford and 2D quantification kits. The recovered protein diluted in 2D rehydration buffer was separated either by one-dimensinal mini-gel electrophoresis or two-dimensional gel electrophoresis (2D) followed by ImageMaster software analysis. Results Protein quantification protocols by Bradford and 2D were compatible with 2D rehydration buffer. With the exception of the protein recovery rates for the ultracentrifugation and ammonium sulphate precipitation which were 40% and 4%, respectively, the protein recovery rates for remained methods were 22%. In view of protein spot boundary, number and resolving ability, aectone-precipitated protein produced the best quality of 2D images, followed by precipitation protocols of ethanol and chloroform/methanol, and ultracentrifugation. Acidified acetone/methanol protocol produced the greatest protein spot lost. Conclusion Acetone precipitation protocol is feasible with protein samples with high concentration and small volume, while ultracentrifugation is for other protein samples.