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A biocompatible fluorescent nanoprobe for singlet oxygen (¹O₂) detection in biological systems was designed, synthesized, and characterized, that circumvents many of the limitations of the molecular probe Singlet Oxygen Sensor Green^® (SOSG). This widely used commercial singlet oxygen probe was covalently linked to a polyacrylamide nanoparticle core using different architectures to optimize the response to ¹O₂. In contrast to its molecular counterpart, the optimum SOSG‐based nanoprobe, which we call NanoSOSG, is readily internalized by E. coli cells and does not interact with bovine serum albumin. Furthermore, the spectral characteristics do not change inside cells, and the probe responds to intracellularly generated ¹O₂ with an increase in fluorescence.