Ginsenoside Rg1, a natural triterpenoid saponins compound isolated from the Panax species, has been found to possess neuroprotective properties in neurodegenerative diseases such as Alzheimer's disease (AD). However, its pharmacological mechanism on AD has not been studied. In this study, an ultra-performance liquid chromatography combined with quadrupole time of-flight mass spectrometry (UPLC-Q/TOF-MS) based non-targeted metabolomics strategy was performed to explore the mechanism of Ginsenoside Rg1 protecting against AD mice by characterizing metabolic biomarkers and regulation pathways changes. A total of nineteen potential metabolites in serum were discovered and identified to manifest the difference between wild-type mice and triple transgenic mice in control and model group, respectively. Fourteen potential metabolites involved in ten metabolic pathways such as linoleic acid metabolism, arachidonic acid metabolism, tryptophan metabolism and sphingolipid metabolism were affected by Rg1. From the ingenuity pathway analysis (IPA) platform, the relationship between gene, protein, metabolites alteration and protective activity of ginsenoside Rg1 in AD mice are deeply resolved, which refers to increased level of albumin, amino acid metabolism and molecular transport. In addition, quantitative analysis of key enzymes in the disturbed pathways by proteomics parallel reaction was employed to verify changed metabolic pathway under Ginsenoside Rg1. The UPLC-Q/TOF-MS based serum metabolomics method brings about new insights into the pharmacodynamic studies of Ginsenoside Rg1 on AD mice.