Protein preparation is a key step in two-dimensional electrophoresis(2-DE)analysis.In order to establish a suitable protein preparation method for Porphyra haitanensis thalli,three sample preparation methods of plant,trichloroacetic acid/acetone precipitation(TCA/ACE)method,urea/thiourea extraction(UREA/THI)method and phenol extraction methanol/ammonium acetate precipitation method,and three protein lysis buffers were compared.The results showed that the optimal method of proteins preparation of P.haitanensis thalli was extracted using TCA/ACE method and dissolved with the lysis buffer C(7 mol/L urea,2 mol/L thiourea,2%CHAPS,2%TritonX-100,2% IPG Buffer pH 3-10,65 mmol/L DTT).Using this method,the yield was the highest and 2-DE map has the most spots,clear backgrounds,high resolution and great repeatability.At the same time,we also found that using the IPG strips(pH 4-7)in 2-DE analysis can extract the protein of P.haitanensis thalli better than IPG strips(pH 3-10).