Glyphosate (GlyP) is a synthetic herbicide widely used around the world alone or together with its analog glufosinate (GluF). GluF, GlyP and its degradation product, aminomethylphosphonic acid (AMPA) have been detected in numerous foodstuffs and environmental water sources, indicating these potential harmful compounds may come into contact with humans, therefore, it is urgent to develop simple and sensitive techniques to detect them. PURPOSE: To develope a powerful technique for the detection of GluF, GlyP and AMPA residues in local surface water system with flow-gated capillary electrophoresis (CE). METHODS: Firstly, Surface Water samples were first fluorogentically derivatized with 4-Fluoro-7-nitrobenzofurazan (NBD-F) in a low-conductivity buffer at room temperature. Secondly, the sample mixture was injected electroosmotically into a separation capillary while negatively charged analytes are electrophoretically rejected. Thirdly, a reversal voltage was applied to push out injected sample while analytes were concentrated at the boundary between running buffer and sample medium based on the field-amplified sample injection (FASI) coupled with electrokinetic supercharging (EKS). Lastly, concentrated analytes were detected with laser induced fluorescence detector. RESULTS: The LODs were estimated to be 50.0 pM, 5.0 pM, and 10.0 pM for GluF, AMPA, and GlyP, respectively. The sensitivity improvement increased up to 296, 444, and 861 folds for GluF, AMPA, and GlyP, respectively. CONCLUSION: This method showed great potential for trace quantitative analysis of negatively charged herbicide residues in local water system. It could also be used in the analysis of other samples including biological fluids after proper sample preparation procedures such as solid phase extraction.