Background: The National Institute on Aging-Alzheimer's Association (NIA-AA) has proposed a biological definition of Alzheimer's disease (AD): individuals with both abnormal amyloid and tau biomarkers (A+T+) would be defined as AD. It remains unclear why different cognitive status is present in subjects with biological AD. Resting-state functional magnetic resonance imaging (rsfMRI) has provided an opportunity to reveal the brain activity patterns in a biologically-defined AD cohort. Accordingly, we aimed to investigate distinct brain activity patterns in subjects with existed AD pathology but in the different cognitive stages. Method: We selected individuals with AD pathology (A+T+) and healthy controls (HC, A−T−) based on the cerebrospinal fluid (CSF) biomarkers. According to the cognitive stage, we divided the A+T+ cohort into three groups: (1) preclinical AD; (2) prodromal AD; and (3) AD with dementia (d-AD). We compared spontaneous brain activity measured by a fractional amplitude of low-frequency fluctuation (fALFF) approach among four groups. Results: The analysis of covariance (ANCOVA) results showed significant differences in fALFF in the posterior cingulate cortex/precuneus (PCC/PCu). Further, compared to HC, we found increased fALFF values in the right inferior frontal gyrus (IFG) in the preclinical AD stage, whereas prodromal AD patients showed reduced fALFF in the bilateral precuneus, right middle frontal gyrus (MFG), right precentral gyrus, and postcentral gyrus. Within the dAD group, both hyperactivity (right fusiform gyrus, right parahippocampal gyrus (PHG)/hippocampus, and inferior temporal gyrus) and hypoactivity (bilateral precuneus, left posterior cingulate cortex, left cuneus and superior occipital gyrus) were detected.