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Serotonin (5-hydroxytryptamine, 5-HT) is a pivotal monoamine neurotransmitter, which is widely distributed in human brain for biological, physical and psychopathological processes. The content of 5-HT can support diagnose of various diseases. To selectively detect 5-HT is very important in clinical medicine. Here, a novel microbiosensor for 5-HT is studied on acupuncture needle. Molecularly imprinted film enwrapped 5-HT was electropolymerized onto bimetallic gold/platinum (Au/Pt) nanoparticles on acupuncture needle microelectrode (ANME). Au/Pt nanostructure exhibited active sites to catalyze the oxidation of 5-HT and bind the generated polymer. 5-HT can be enwrapped by the functional monomer of pyrrole (Py) in the process of electropolymerization with suitably electroactive conformation. Comparing with interfaces of single metal or molecularly imprinted layer, synergistic microbiosensor exhibit better performance for 5-HT. 5-HT can be adsorbed and catalytically oxidized by the imprinted cavities. Under optimized conditions, the peak current linearly increases with the concentration of 5-HT from 0.03 to 500 mu M, and a detection limit of 0.0106 mu M is obtained. The performance of this microbiosensor is competitive with previous studies. Furthermore, the prepared microbiosensor showed effective application to analyze 5-HT in human serum and urine. Interestingly, the microbiosensor expressed the real-time monitoring ability to 5-HT from stimulated PC12 cells by K+. The microbiosensor also exhibited high selectivity, stability and reproducibility, which is promising in view of the low price, fast response and simple operation.