The Free Flow Electrophoresis micro Chip with voltage applied in two-dimension and coupled with hydraulic control unit was designed in this paper, which can achieve fluid transport and sample separation in the electrophoresis process. The Alpha gel scanning imaging system was used as the detector, which could observe experiment phenomenon, record and analyse experiment results. Under the optimized conditions of the 10mmol borate buffer contain 3 %( v/v) glycerol, at pH 10 and the ratio of field strength in two-dimension at 1:5, the mixed amino acids including phenylalanine (L-Phe) and lysine (L-Lys) were successfully separated in the μ-FFE analytical system. The experiment had shown that the correlation coefficient of the L-Phe and L-Lys was up to 0.993 and 0.992, respectively. The results illustrated that μ-FFE have showed great potential perspective in the separation of mix Amino Acids, especially in the quantitative test of the trace Amino Acids in clinic samples.