Postoperative cognitive dysfunction (POCD) is a subtle cognitive dysfunction, especially memory impairment for weeks or months after surgery. The underlying pathophysiological mechanism of POCD is still unclear. The aim of this study was to exploratively investigate the potential mechanism of POCD by identifying the differences among metabolic profiles of control rats, POCD and no-POCD rats after isoflurane anesthesia based on GC-MS, and subsequently discovering POCD biomarkers. In this paper, a feature-variable selection method, subwindow permutation analysis (SPA), was employed to seek the key metabolites distinguishing POCD from control group, POCD from no-POCD group. Fortunately, two key metabolites, hexadecanoic acid and myo-Inositol, were both screened out for discriminating POCD and control, POCD and no-POCD rats. It suggested that they may reveal the disturbances between POCD and control, POCD and no-POCD rats, which may be the potential biomarkers of POCD. Furthermore, related possible pathogenesis was taken into account on the basis of the relevant literatures and pathway databases. It suggested that POCD was probably related to disturbed hexadecanoic acid metabolism and myo-Inositol metabolism. All the results demonstrated that the proposed metabolic profiling approach and SPA method may be effective for exploring metabolic perturbations and possible biomarkers for POCD.