Abstract: To establish an HPLC-FLD method for the investigation of monoamine neurotransmitters and related substances in mouse frontal cortex，hippocampus，striatum，and hypothalamus.Mouse brain samples were deproteinized by perchloric acid and analyzed.The separation was carried out on a Lichrospher C₁₈ column(250 mm×4.6 mm，5 μm).The mobile phase was composed of pH 3.06 buffer(including 1 mmol/L heptanesulfonic acid sodium salt，30 mmol/L NaH₂PO₄-H₃PO₄ solution) and methanol in the ratio of 85∶15；the flow rate was 1.0 mL/min and the column temperature was 35 °C.Fluorescence detection：the excitation and emission wavelengths were set at λ_ex=280 nm，λ_em=315 nm.Under the optimized conditions，norepinephrine(NE)，dopamine(DA)，5-hydroxytryptamine (5-HT)，3，4-dihydroxy-phenyl acetic acid(DOPAC)，5-hydroxyindoleacetic acid(5-HIAA)，tyrosine(Tyr)，and tryptophan(Trp) in mouse brain were detected.NE，DA，and 5-HT were quantified.The linearity ranges of NE，DA， and 5-HT were 2.4-471.0 ng/mL(r=0.999 6)，2.6-519.5 ng/mL (r=0.999 4)，and 2.4-469.3 ng/mL(r=0.999 1)，respectively.The intra- and inter-day precision was always lower than 9.0%. The accuracy could attain the demands and the absolute recovery values were always higher than 90%. The proposed method has good specificity and accuracy.It could be used to analyze the content differences of monoamine neurotransmitters and related substances in different mouse brain areas.