As a novel nano-technology, nano-scale channel electrophoresis separation (NCS) technique (a hole or channel 0.1-500 nanometer in diameter) is widely applied in biomedical, chemical fields, and so on. Multiphoton-excited fluorescence (MPEF) takes advantages of high spatial resolution (sub-fL level) and high sensitivity (single molecule). Especially, MPEF only occurs in an ultra-low volume (about 100 aL) in the vicinity of the focus, which is perfectly adequate for NCS. In order to provide a new method for an effective separation and detection of biological components, MPEF experiment has been conducted with three fluorescent molecular of different capillary sizes, the results showed that multiphoton-excited volume did not relate to the dimension of capillary. Based on that, NCS was coupled with MPEF detection to analyze amino acids, and 21 amino acids mixture tagged with fluorescein isothiocyanate have been completely separated and sensitively detected. Nine amino acid samples at different concentrations were determined with NCS-MPEF, the calibration curve for each amino acid was established with the linear dynamic ranges above two orders of magnitudes and the linear correlation coefficients γ > 0.99. It was shown that the system was qualified for the analysis of amino acids. Furthermore, the free amino acids in tobacco leaf were successfully analyzed. NCS-MPEF takes the advantages of aL-level excitation volume and nano-scale channel electrophoresis separation and could be further applied to complex sample analysis.