Cytochrome c (Cytc) is a key redox protein for energy metabolism and apoptosis in cells. The activation of Cytc is composed of several steps, including its transfer to the mitochondrial membrane, binding to cytochrome c heme lyase (CCHL) and covalent attachment to heme. The spectroscopic methods are often applied to study the structural changes of Cytc. However, they require the isolation of Cytc from cells and have limited availability under physiological conditions. Despite recent studies to elucidate the tightly regulated folding mechanism of Cytc, the role of these events and their association with different conformational states remain elusive. Here, we provide a genetically encoded fluorescence method that allows monitoring of the conformational changes of Cytc upon binding to heme and CCHL. Cerulean and Venus fluorescent proteins attached at the N and C terminals of Cytc can be used to determine its unfolded, intermediate, and native states by measuring FRET amplitude. We found that the noncovalent interaction of heme in the absence of CCHL induced a shift in the FRET signal, indicating the formation of a partially folded state. The higher concentration of heme and coexpression of CCHL gave rise to the recovery of Cytc native structure. We also found that Cytc was weakly associated with CCHL in the absence of heme. As a result, a FRET-based fluorescence approach was demonstrated to elucidate the mechanism of heme-induced Cytc conformational changes with spatiotemporal resolution and can be applied to study its interaction with small molecules and other protein partners in living cells.