Objective To observe the learning and memory ability of rats after injection of Aβ_25-35 protein in different concentrations into the lateral ventricle assessed by Morris water maze test, and to explore the optimal concentration of Aβ_25-35 in the preparation of AD model rats. Methods Male SD rats were randomly divided into sham operated group and model group. The rats of model group received Aβ_25-35 injection in concentrations of 2 μg/μL, 4 μg/μL and 8 μg/μL, respectively. According to the Rat Brain Stereotaxic Atlas, 5 μL of aggregation of Aβ_25-35 was injected into the right lateral ventricle to establish the AD rat model. 7 days after successful modeling, Morris water maze was used to test thechanges of learning and memory ability of the rats. Results There was no significant difference in the average swimming speed between the two groups (P> 0.05).The escape latency time of rats in the model group was significantly increasedcompared with the sham group (P< 0.05).In the model group, the escape latency time of rats treated with 4 μg/μL and 8 μg/μL Aβ_25-35 was significantly increased compared with the rats injected with 2 μg/μL (P< 0.05), while there was no significant difference between rats treated with 4 μg/μL and 8 μg/μL Aβ_25-35 (P> 0.05). The activity time and distance of target quadrant of the rats injected with different concentration of Aβ_25-35in the model group were significantly reduced compared with the sham group (P< 0.05), but no significant difference amongthe rats treated with different Aβ_25-35 concentrations (P> 0.05). Compared with the sham-operated group, the number of platform-crossing of rats injected with different doses of Aβ_25-35in the model group were significantly reduced (P< 0.05). In the model group, the rats treated with 4 μg/μL and 8 μg/μL was significantly reduced compared with the group with 2 μg/μL injection (P< 0.05). There was no significant difference between the rats injected with 4 μg/μL and 8 μg/μL (P> 0.05). Conclusions The recommended dose and concentration of Aβ_25-35 to be injected into the unilateral ventricle to establisha rat model of Alzheimer's disease is 4 μg/μL in a volume of 5 μL.