Oxidative stress is important for the etiology and pathogenesis of Alzheimer's disease (AD). Research tools that can conveniently evaluate oxidative stress in AD models are expected to catalyze and accelerate research on AD. This study explored the use of genetically encoded fluorescent indicators (GEFIs) to detect mitochondrial oxidative stress in organotypic brain slices and AD mouse models. To enable ratiometric normalization and avoid tissue autofluorescence, we genetically fused a green fluorescent hydrogen peroxide (HO) indicator, HyPer7, with each of two selected, bright red fluorescent proteins (RFPs), mScarlet-I and tdTomato. The resultant indicators, namely, HyPerGR and HyPerGR, were tagged with mitochondrial targeting sequences and examined for localization and function in cultured HeLa cells and primary mouse neurons. We further utilized HyPerGR, which is a genetic fusion of HyPer7 with tdTomato, to monitor mitochondrial HO in response to the human β-amyloid 1-42 isoform (Aβ) in cultured brain slices and an AD mouse model. Owing to the high sensitivity and low autofluorescence interference resulting from HyPerGR, we successfully detected Aβ-mediated mitochondrial HO in these AD models. The results suggest that HyPerGR is a valuable tool for studying mitochondrial oxidative stress in tissues and animals.