Long-term or excessive application of morphine leads to tolerance and addiction, which hindered its conventional applications as a drug. Although tremendous progress has been made on the mechanisms of morphine, crucial evidence elaborating the neurobiological basis of tolerance and dependence is still lacking. To further explore the physiological adaptions during morphine's application, a systematic screening of small molecules in blood has been carried out. The plasma of morphine dependent rats was collected at different time points with or without naloxone treatment, and was analyzed by gas chromatography-mass spectrometry (GC-MS). Partial least squares discriminate analysis (PLS-DA) and the Student's t Tests with the false discovery rate (FDR) correction were conducted on the normalized data for the distinction of groups and the identification of the most contributed metabolites. Clear separation is observed between different treatments, and 29 out of 41 metabolites changes significantly compared with the corresponding controls. The concentration of threonine, glycine, serine, beta-d-glucose and oxalic acid are consistently changed in all morphine treated groups compared with controls. Through this experiment we find characteristic metabolites in different dependent states and discuss the possible compensation effects. The interpretation of these metabolites would throw light on the biological effects of morphine and reveal the possibilities to become marker of morphine addiction.