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A quantitative determination of six neuroactive amino acids (NAAs) was performed by capillary zone electrophoresis with amperometric detection (CZE-AD). This CZE-AD method utilized two electrolytes: the borate solution flowing in a capillary has the NAAs-separation effects, and the sodium hydroxide (NaOH) solution filled in the detection reservoir for the amperometric analysis of NAAs. The following experimental parameters were optimized: the working electrode potential, the pH value, the component, and the concentration of running buffer, the separation voltage, and the injection time on CZE-AD. Then, under the optimum conditions, the six NAAs could be completely separated in 30 min and had well-shaped AD responses at 0.75 V (versus SCE) on a copper electrode. The linear calibration range of NAAs was from 5 × 10−4 to 5 × 10−6 mol L−1 with the limits of detection (LODs) ranging from 10−6 to 10−7 mol L−1 (signal-to-noise ratio = 3), and the relative standard deviations (RSDs) of the migration time and peak area were 0.45–0.55 and 3.8–6.3 %, respectively. Moreover, this method has succeeded in human serum analysis, and the determined contents of the six NAAs in human serum were in an average recovery range of 85.3–117.9 %, which confirmed the validity and practicability of this method.