Abstract: To investigate potential mechanism of nicotine dependence and evaluate the monoamine neurotransmitter metabolism caused by nicotine as a function of time, online microdialysis sampling coupled with high performance liquid chromatography and electrochemical detector was developed and applied to determine the contents of dopamine (DA), 3, 4-dihydroxyphenyl acetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxylindole acetic acid (5-HIAA) in the brain striatum of live rats after nicotine injection. Striatum dialysates were collected every 15 min at a flow rate of 2.0 μL/min and separated immediately on a HS F5 column under the gradient elution condition. The results showed that the linear range of the method were from 0.2 to 500.0 ng/mL, the recoveries ranged from 86.2% to 105.9%, while the intra-day and inter-day precisions were no more than 9.7%. In vitro microdialysate recoveries of DA, DOPAC, HVA, 5-HT and 5-HIAA ranged from 25.3% to 28.3%. After intraperitoneal injection of nicotine at a rate of 2.0 mg/kg, the release of DA and 5-HT in brain striatum of rat did not increase continuously, while the release of HVA and 5-HIAA rose significantly. The method is simple, fast, sensitive and suitable for the dynamic analysis of monoamine neurotransmitters in brain of live animal administrated by nicotine via digestion.