<p>Extracellular molecules such as ions, amino acids, nucleotides, and proteins are essential mediators of intercellular communication in multicellular organisms. These diffusible factors contribute to development and homeostasis of organisms by coordinating signaling among distant cells. Despite the widespread use of fluorescent indicators for studying intracellular signaling, introducing genetically encoded indicators often complicates the control of their subcellular localization, making it difficult to distinguish intracellular from extracellular signals. Consequently, monitoring the spatiotemporal dynamics of diffusible molecules in the extracellular space has remained technically challenging. Here we report a versatile chemical anchoring method using lipid–polyethylene glycol (PEG) conjugates that immobilizes fluorescent protein (FP)–based indicators on the extracellular surface of living cells. This approach enables stable localization of FP-based indicators on hippocampal neurons in both primary cultures and in acute brain slices. Lipid–PEG–anchored indicator for potassium ion and that for glutamate allowed real-time optical monitoring of ion and neurotransmitter release from neurons during spontaneous and electrically evoked neuronal activity. This non-genetic labeling strategy provides localized, rapidly applicable, high sensitivity, stability, and reproducibility, offering a versatile platform for quantitative monitoring of extracellular signaling events near membranes of living cells in a low invasive manner.</p>