As an important biomarker for the early stage of apoptosis, cytochrome c (Cyt c) has been recognized as a key component of the intrinsic apoptotic pathway. Fluorescence imaging tools enabling detection of Cyt c in apoptotic signaling have been rarely explored, though they are critical for cell biology and clinical theranostics. Here, we designed a novel label-free N-doped carbon dot (N-doped CD)-based nanosensor that enables fluorescence activation imaging of Cyt c release in cell apoptosis. The inner filter effect of Cyt c towards N-doped CDs enabled quantitative Cyt c measurement. The nanosensor exhibited high sensitivity and selectivity, rapid response, good cell-membrane permeability and low cytotoxicity. All these features are favorable for in situ visualization of Cyt c for apoptosis research. Notably, the developed nanosensor was successfully applied to monitor intracellular release of Cyt c, and to visualize Cyt c in living zebrafish for the first time. Moreover, it also provided a viable platform for cell-based screening of apoptosis-inducing compounds. In virtue of these advantages and potential, the developed assay not only holds great significance for the better understanding of certain diseases at the cellular level, but also provides an invaluable platform for apoptotic studies and screening of anti-cancer drugs toward drug development.