Alzheimer’s disease (AD) is a growing global health crisis, with annual per-patient management costs exceeding USD 50,000 and cases projected to surpass 150 million worldwide by 2050. The urgent need for innovative, clinically translatable diagnostic platforms has driven the adoption of advanced nanotechnology-based approaches. Here, we present a label-free, multispectral fluorescence lifetime imaging microscopy (FLIM) platform for real-time, non-invasive, 3D metabolic profiling of live human cerebral organoids (COs) modeling AD. We established two types of COs: (1) inducible organoids derived from hiPSCs with the PSEN1M146I mutation, and (2) patient-derived organoids from familial AD iPSCs. Both models faithfully recapitulate hallmark AD pathologies, including amyloid-β accumulation and neural maturation. FLIM enabled detection of significant alterations in NADH-associated auto-fluorescence lifetimes in AD COs, correlating with increased inflammation and oxidative stress. These intrinsic metabolic signatures serve as robust, label-free biomarkers for AD, offering a non-invasive alternative to current invasive clinical diagnostics. Our molecular imaging platform aligns with recent regulatory shifts prioritizing human-derived models and AI integration, and is adaptable for broad applications in neurodegenerative disease modeling, drug screening, and precision medicine. This work demonstrates the transformative potential of nanotechnology-driven organoid imaging for accelerating AD research, diagnosis, and therapeutic development.