In study, we investigated the effect of treatment with combination of < 3 kDa + Rg1 on scopolamine-induced cognitive impairment in mice and the mechanism of BDNF/TrKB/CREB signaling pathway in PC12 cells is regulated. 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 H&E, Nissl, and Golgi staining. Additionally, immunohistochemistry showed that the < 3 kDa+Rg1 treatment significantly decreased AChE activity and increased ChAT content in the hippocampus. The treatment upregulated 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 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.