Hair is a noninvasive valuable biospecimen for the long-term assessment of endogenous metabolic disturbance. Whether the hair is suitable for identifying biomarkers of the Alzheimer’s disease (AD) process remains unknown. We aim to investigate the metabolism changes in hair after β-amyloid (Aβ1-42) exposure in rats using ultra-high-performance liquid chromatography-high-resolution mass spectrometry–based untargeted and targeted methods. Thirty-five days after Aβ1-42induction, rats displayed significant cognitive deficits, and forty metabolites were changed, of which twenty belonged to three perturbed pathways: (1) phenylalanine metabolism and phenylalanine, tyrosine, and tryptophan biosynthesis—l-phenylalanine, phenylpyruvate, ortho-hydroxyphenylacetic acid, and phenyllactic acid are up-regulated; (2) arachidonic acid (ARA) metabolism—leukotriene B4 (LTB4), arachidonyl carnitine, and 5(S)-HPETE are upregulation, but ARA, 14,15-DiHETrE, 5(S)-HETE, and PGB2 are opposite; and (3) unsaturated fatty acid biosynthesis— eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), FA 18:3 + 1O, and FA 18:3 + 2O are downregulated. Linoleic acid metabolism belonging to the biosynthesis of unsaturated fatty acid includes the upregulation of 8-hydroxy-9,10-epoxystearic acid, 13-oxoODE, and FA 18:2 + 4O, and downregulation of 9(S)-HPODE and dihomo-γ-linolenic acid. In addition, cortisone and dehydroepiandrosterone belonging to steroid hormone biosynthesis are upregulated. These three perturbed metabolic pathways also correlate with cognitive impairment after Aβ1-42stimulation. Furthermore, ARA, DHA, EPA,l-phenylalanine, and cortisone have been previously implicated in the cerebrospinal fluid of AD patients and show a similar changing trend in Aβ1-42rats’ hair. These data suggest hair can be a useful biospecimen that well reflects the expression of non-polar molecules under Aβ1-42stimulation, and the five metabolites have the potential to serve as novel AD biomarkers.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12035-023-03343-6.