In vivo electrochemical analysis is of great significance in understanding the dynamics of various physiological and pathological activities. However, the conventional microelectrodes for electrochemical analysis are rigid and permanent, which comes with increased risks for long-term implantation and secondary surgery. Here, we develop one biodegradable microelectrode for monitoring the dynamics of extracellular Ca2+ in rat brain. The biodegradable microelectrode is prepared by sputtering gold nanoparticles (AuNPs) on a wet-spun flexible poly­(l-lactic acid) (PLLA) fiber for conduction and transduction and coating a Ca2+ ion-selective membrane (ISM) with a PLLA matrix on the PLLA/AuNPs fiber, forming PLLA/AuNPs/Ca2+ISME (ISME = ion-selective microelectrode). The prepared microelectrode shows excellent analytical properties including a near-Nernst linear response toward Ca2+ over the concentration range from 10 μM to 50 mM, good selectivity, and long-term stability for weeks as well as biocompatibility and biodegradability. The PLLA/AuNPs/Ca2+ISME can monitor the dynamics of extracellular Ca2+ following spreading depression induced by high potassium even if in the fourth day. This study provides a new design strategy for the biodegradable ISME and promotes the development of biodegradable microelectrodes for long-term monitoring of chemical signals in brain.