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A novel series of profluorescent rhodamine nitroxide conjugates are synthesized utilizing well‐known isonitrile‐based multicomponent reactions (IMCRs). The synthesized conjugates are rationally designed as mitochondria‐targeting probes for the detection of reactive oxygen species in living cells. Herein, the synthesized probes demonstrate high selectivity to target the mitochondria of both of PC3‐ and NIH3T3‐cells which represent cancer and normal cell lines. Attaching TEMPO nitroxide to rhodamine leads to fluorescence quenching, allowing for ROS detection and quantification. The prepared sensors provide a reliable method for distinguishing between different oxidative environments in living organisms through different levels of fluorescence to be measured. The use of the Ugi multicomponent reaction enables an efficient and versatile synthetic approach, offering significant advantages over previously reported methods for constructing ROS‐detecting probes. The simplicity of the reaction setup and the ability to generate a diverse library of products by varying Ugi components make this protocol highly adaptable for further chemical modification and potential applications in biological systems.