Objective To analyze the different expressed protein of pleural effusion caused by tuberculosis and to identify proteins associated with tuberculous pleural effusion for building an economic , rapid, and accurate diagnostic method. Methods Two-dimensional gel electrophoresis technology was applied to separate protein in pleural effusion of 20 cases of tuberculous pleurisy ,19 cases of lung cancer patients and 6 cases of transudate. Analysis of isoelectric point, the range of molecular weight, matching rate and grey value of the protein was carried out by the PDQuest8.0 software.Then the electrophoregram was compared to get the distinct protein. Results There were 13 differential protein spots between tuberculous pleural effusion and the transudateand 9 protein spots were highly expressed for two folds, but 4 protein spots poorly expressed for 0.5 folds in pleural effusion caused by tuberculous pleurisy. There were 11 differential protein spots between tuberculous and malignant pleural effusion and 5 protein spots were highly expressed , but 4 protein spots poorly expressed in pleural effusion caused by tuberculous pleurisy , while 2 protein spots were expressed only in the pleural effusion caused by lung cancer. Conclusion Two-dimensional electrophoresis technology is available to acquire an electrophoretogram of pleural effusion caused by tuberculosis , lung cancer and transudate with well resolution and high repetition rate.In addition, there are different protein spots.