Vitamin E plays an exemplary role in living organisms. α-Tocopherol is the most superior and active form of naturally occurring vitamin E that meets the requirements of human beings as it possesses the α-tocopherol transfer protein (α-TTP). α-Tocopherol deficiency can lead to severe anemia, certain cancers, several neurodegenerative and cardiovascular diseases, and most importantly male infertility. As a result of the depletion of its natural sources, researchers have tried to employ metabolic engineering to enhance α-tocopherol production to meet the human consumption demand. However, the metabolic engineering approach relies on the metabolic flux of a metabolite in its biosynthetic pathway. Analysis of the metabolic flux of a metabolite needs a method that can monitor the α-tocopherol level in living cells. This study was undertaken to construct a FRET (fluorescence resonance energy transfer)-based nanosensor for monitoring the α-tocopherol flux in prokaryotic and eukaryotic living cells. The human α-TTP was sandwiched between a pair of FRET fluorophores to construct the nanosensor, which was denoted as FLIP-α (the fluorescence indicator for α-tocopherol). FLIP-α showed excellence in monitoring the α-tocopherol flux with high specificity. The sensor was examined for its pH stability for physiological applications, where it shows no pH hindrance to its activity. The calculated affinity of this nanosensor was 100 μM. It monitored the real-time flux of α-tocopherol in bacterial and yeast cells, proving its biocompatibility in monitoring the α-tocopherol dynamics in living cells. Being noninvasive, FLIP-α provides high temporal and spatial resolutions, which holds an indispensable significance in bioimaging metabolic pathways that are highly compartmentalized.