In study, we investigated the effect of treatment with combination of walnut peptides with molecular weight < 3 kDa and ginsenoside Rg1 (< 3 kDa + Rg1) on scopolamine-induced cognitive impairment in mice and the mechanism of brain-derived neurotrophic factor (BDNF)/tyrosine kinase B (TrKB)/cAMP response element-binding protein (CREB) signaling pathway in PC12 cells. In behavioral experiments, < 3 kDa + Rg1 treatment improved the memorizing ability of mice. Treatment with < 3 kDa + Rg1 significantly regulated the function of neurotransmitters and effectively improved the morphology of the neurons determined by hematoxylin and eosin (H&E), Nissl, and Golgi staining. Additionally, immunohistochemistry showed that the < 3 kDa + Rg1 treatment significantly decreased acetylcholinesterase (AChE) activity and increased choline acetyl transferase (ChAT) content in the hippocampus. The treatment upregulated vesicular acetylcholine transporter (VAChT), activated the BDNF/TrKB/CREB signaling pathway, improved the remodeling of dendritic spines, and enhanced cholinergic functions. In the scopolamine-induced PC12 cells, combination treatment increased thioredoxin-1 (Trx-1) expression after administering TrKB and activated signaling pathway. The results showed combination of < 3 kDa + Rg1 activated the BDNF/TrKB/CREB signaling pathway by regulating function of neurotransmitters and enhanced cholinergic function to decrease cognitive impairment.