Abstract Cholinergic innervation in brain is involved in modulating neurovascular function including cerebral blood flow hemodynamics in response to neuronal activity. Cholinergic deficit is associated with pathophysiology in Alzheimer’s disease, albeit the etiology remains to clarify. In current study, neocortex cerebral blood flow response to acetylcholine was evaluated by Laser-Doppler Flowmetry in 3xTgAD Alzheimer’s disease model) and wild type mice of two age groups. The peak of cerebral blood flow to acetylcholine (i.v.) from baseline levels (% ΔrCBF) were higher in young 3xTgAD vs. in wild type mice (48.35; CI:27.03-69.67 vs. 22.70; CI:15.5-29.91, p < 0.05); this was reversed in old 3xTgAD mice (21.44; CI:2.52-40.35 vs. 23.25; CI:23.25-39). Choline acetyltransferase protein was reduced in neocortex, while cerebrovascular reactivity to acetylcholine was preserved in young 3xTgAD mice. This suggests of endogenous acetylcholine deficit and possible cholinergic denervation from selected cholinergic nuclei within basal forebrain. The early deposition of tauopathy moieties (mutant hTau and pTau181) and its coincidence in cholinergic cell clusters, were observed occasionally at basal forebrain of 3xTgAD mice including substantia innominate, nucleus Basalis of Meynert and nucleus of horizontal limb diagonal band of Broca. A prominent feature was microglia interacting tauopathy and demonstrated a variety of morphology changes particularly when located in proximity to tauopathy. The microglia ramified phenotype was reduced as evaluated by the ramification index and Fractal analysis. Increased microglia senescence, identified as SASP (senescence-associated secretory phenotype), was colocalization with p16Ink4ɑ, a marker of irreversible cell cycle arrest in old 3xTgAD vs. wild type mice (p = 0.001). The p16Ink4ɑ was also observed in neuronal cells bearing tauopathy within basal forebrain of 3xTgAD mice. TNF-ɑ, the pro-inflammatory cytokine elevated persistently in microglia (Peason’s correlation coefficient = 0.62), and the loss of cholinergic cells in vulnerable basal forebrain environment was indicated by image analysis in 3xTgAD mice, which linked to the cholinergic deficits in neocortex rCBF hemodynamics. Our study revealed the early change of CBF hemodynamics to acetylcholine in 3xTgAD model. As a major effector of brain innate immune activation, microglia SASP with age related disease progression is indicative of immune cell senescence, which contributes to chronic inflammation and cholinergic deficits at basal forebrain. Targeting neuroinflammation and senescence may mitigate cholinergic pathophysiology in Alzheimer’s disease.