AbstractDescribed here is that Au−C≡C bonds showed the highest stability under biological conditions, with abundant thiols, and the best electrochemical performance compared to Au−S and Au−Se bonds. The new finding was also confirmed by theorical calculations. Based on this finding, a specific molecule for recognition of Fe2+ was designed and synthesized, and used to create a selective and accurate electrochemical sensor for the quantification of Fe2+. The present ratiometric strategy demonstrates high spatial resolution for real‐time tracking of Fe2+ in a dynamic range of 0.2–120 μM. Finally, a microelectrode array with good biocompatibility was applied in imaging and biosensing of Fe2+ in the different regions of live mouse brains. Using this tool, it was discovered that the uptake of extracellular Fe2+ into the cortex and striatum was largely mediated by cyclic adenosine monophosphate (cAMP) through the CREB‐related pathway in the brain of a mouse with Alzheimer's disease.