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Phagocyte respiratory burst in immune responses generates enormous amounts of reactive oxygen species (ROS) to fulfill primary defense against neoplasia. However, the beneficial functions associated with ROS, especially the potent oxidant/nucleophile peroxynitrite, in an immunological process are still ambiguous. Herein, we report the construction and biological assessment of cyanine-based fluorescent biosensors, which are based on a nonoxidative strategy for peroxynitrite detection. The established nonoxidative strategy is composed of nucleophilic substitution and nanoaggregate formation initiated by peroxynitrite. The proposed nonoxidative strategy in this study could maintain cellular oxidative stress in the critical process of detection and preserve homeostasis of cell metabolism. The remarkable detection sensitivity, reaction selectivity, and spectral photostability of our biosensors enabled us to visualize endogenous peroxynitrite levels in immune-stimulated phagocytes. With the aid of basal peroxynitrite imaging in an acute peritonitis model, the visualization of peroxynitrite level variations in immune responses of tumorigenesis was accomplished assisted by our biosensors. It is envisioned that our strategy provides a promising tool for early tumor diagnosis and evaluation of tumor suppression in the process of immune responses without disturbing the functions of ROS signaling transduction.