Cellular perturbations underlying Alzheimer’s disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes—which we refer to as the early cortical amyloid response—were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with β-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis. Display omitted •Single-nucleus profiling of human cortex biopsies uncovers amyloid-associated states•Upper-layer pyramidal neurons show hyperactivity prior to degeneration•Microglial states correlate with pathological and clinical progression•Signatures of amyloid production identified in both neurons and oligodendrocytes Generating single-nucleus atlas from cortical biopsies of living individuals at early stage of Alzheimer’s disease, cell states of neurons, microglia, and oligodendrocytes associated with AD pathology are identified.