This study presents a novel, patient-specific 3D microglia-like cell model (MDMi) derived from monocytes, which is more physiologically relevant and experimentally flexible compared to existing models. The MDMi models were generated using a 3D Matrigel-based culture system and co-cultured with neuro-glial cells (ReNeCell VM). Single-cell RNA sequencing (scRNAseq) analysis revealed that 3D MDMi exhibited a more branched morphology and longer survival compared to 2D cultures. The 3D co-culture model showed enhanced microglial-like features and altered cell-to-cell interactions, growth factor secretion profiles, and responses to amyloid-β. Patient-derived MDMi models from Alzheimer's disease (AD) patients and healthy controls were characterized, and their cytokine responses to anti-inflammatory drugs (dasatinib and spiperone) were profiled. The results demonstrate that AD MDMi in 3D co-culture exhibited disease-specific phenotypes, including altered cell-to-cell interactions, secretory profiles, and behaviors in the presence of Aβ aggregates. The study highlights the potential of these patient-specific 3D MDMi models for improving personalized drug development strategies in Alzheimer's disease.This study presents a novel, patient-specific 3D microglia-like cell model (MDMi) derived from monocytes, which is more physiologically relevant and experimentally flexible compared to existing models. The MDMi models were generated using a 3D Matrigel-based culture system and co-cultured with neuro-glial cells (ReNeCell VM). Single-cell RNA sequencing (scRNAseq) analysis revealed that 3D MDMi exhibited a more branched morphology and longer survival compared to 2D cultures. The 3D co-culture model showed enhanced microglial-like features and altered cell-to-cell interactions, growth factor secretion profiles, and responses to amyloid-β. Patient-derived MDMi models from Alzheimer's disease (AD) patients and healthy controls were characterized, and their cytokine responses to anti-inflammatory drugs (dasatinib and spiperone) were profiled. The results demonstrate that AD MDMi in 3D co-culture exhibited disease-specific phenotypes, including altered cell-to-cell interactions, secretory profiles, and behaviors in the presence of Aβ aggregates. The study highlights the potential of these patient-specific 3D MDMi models for improving personalized drug development strategies in Alzheimer's disease.