To select differentially expressed proteins in hippocampus of rats with pattern of Liver Qi stagnation and Spleen deficiency (LQSSD) induced by chronic immobilization stress (CIS), by applying the technique of protein chip and surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). Methods: The rats with pattern of LQSSD according to traditional Chinese medicine (TCM) were replicated by the method of CIS. After twenty-one days’ stress, the protein chip weak cation CM10 and SELDI-TOF-MS were applied in order to do mass spectral analysis of the proteins acquired from the three groups: normal control group, 21 days model group, and Xiaoyaosan (XYS) treatment group. Bio Marker Wizard software were adopted to analyze the differentially expressed proteins. Results: Five differentially expressed proteins were selected between the control group and model group, and they were all highly expressed in the model group. Thirteen one were selected between control group and treatment group, among which 3 were highly expressed in the treatment group, and 10 were highly expressed in the control group. Only 1 differentially expressed protein was selected between the treatment group and the model group and it was highly expressed in the latter. Conclusion: The differentially expressed proteins may be the specific marker proteins in hippocampus of rats with pattern of LQSSD induced by CIS. TCM formula XYS may be an effective intervention for protein expression in hippocampus of rats with pattern of LQSSD.