Thioredoxin (Trx) is one of the major thiol-dependent antioxidants in living systems. The study of Trx functions in redox biology was impeded by the lack of practical tools to track Trx redox dynamics in live cells. Our previous work developed TrxRFP1, the first genetically encoded fluorescent indicator for Trx redox. In this work, we report an improved fluorescent indicator, TrxRFP2, for tracking the redox of Trx1, which is primarily cytosolic and nuclear. Furthermore, because mitochondria specifically express Trx2, we have created a new genetically encoded fluorescent indicator, MtrxRFP2, for the redox of mitochondrial Trx. We characterized MtrxRFP2 as a purified protein and used subcellularly localized MtrxRFP2 to image mitochondrial redox changes in live cells.