Alzheimer's disease (AD) is a common neurodegenerative disease, which is characterized by aberrant accumulation of amyloid-β (Aβ) and neuroinflammation. The purpose of this study was to explore the regulatory effects of long non-coding RNA (lncRNA) MAGI2-AS3 and microRNA-374b-5p (miR-374b-5p) on Aβ-induced neurotoxicity and neuroinflammation, as well as the relationship between MAGI2-AS3 and miR-374b-5p in AD patients. A luciferase reporter assay was used to analyze the interaction between MAGI2-AS3 and miR-374b-5p and between miR-374b-5p and beta-site amyloid precursor protein cleaving enzyme 1 (BACE1). SH-SY5Y and BV2 cells treated with Aβ25–35 were used to mimic neuronal injury and neuroinflammation in AD pathogenesis. Cell viability was evaluated using a MTT assay, and pro-inflammatory cytokine levels were measured using ELISA kits. MAGI2-AS3 and miR-374b-5p expression was examined using quantitative real-time PCR. BACE1 served as a target gene of miR-374b-5p, and MAGI2-AS3 could sponge miR-374b-5p. The expression of MAGI2-AS3 was increased, and miR-374b-5p was decreased in both SH-SY5Y and BV2 cells exposed to Aβ25–35. MAGI2-AS3 reduction enhanced neuronal viability and attenuated neuroinflammation in AD cell models, and miR-374b-5p overexpression led to same effects, but miR-374b-5p inhibition reversed these effects. Serum MAGI2-AS3 and miR-374b-5p levels in AD patients were negatively correlated and correlated with disease severity. The findings indicated that the MAGI2-AS3/miR-374b-5p axis regulates Aβ-induced neurotoxicity in SH-SY5Y cells and neuroinflammation in BV2 cells. The MAGI2-AS3/miR-374b-5p axis may provide novel biomarkers and therapeutic targets for AD. •MAGI2-AS3 sponges miR-374b-5p in both SH-SY5Y and BV2 cells.•MAGI2-AS3 reduction alleviates neurotoxicity and neuroinflammation in AD cell models.•miR-374b-5p mediates the biological function of MAGI2-AS3 in AD cell models.•The MAGI2-AS3/miR-374b-5p axis expression was aberrant in serum of AD patients.