We developed a voltammetric method to determine dopamine (DA) and 5-Hydroxytryptamine (5-HT) selectively and simultaneously in the mouse brain based on the graphene-iron-tetrasulfophthalocyanine (GR-FeTSPc)coated carbon fiber microelectrode (GR-FeTSPc/CFME). The GR-FeTSPc composites possess an improved solubility in aqueous solution and the GR-FeTSPc/CFME exhibits an enhanced electro-catalytic activity towards the oxidation of DA and 5-HT. The reversible redox peaks are observed for DA with the redox peak potentials of 0.15 V and 0.10 V respectively, and only an oxidation peak at 0.36 V for 5-HT can be seen at the GR-FeTSPc/CFME. Differential Pulse Voltammetry (DPV) was used to determinate DA and 5-HT simultaneously, and the detection limits (S/N = 3) were 50 nM for DA and 20 nM for 5-HT respectively. Additionally, the microsensor exhibits good selectivity against AA and homovanillic acid. The experiment results indicate that this microsensor can determine the release of DA and 5-HT simultaneously in the mouse brain on line in vivo and at the same time it can monitor the two biomolecules concentrations changes with time. The research may offer a new method to monitor the release of DA and 5-HT in vivo.