An in-capillary derivatization and stacking capillary electrophoresis (CE) technique has been applied to redeem the detection of dilute analytes in the analysis of gamma-aminobutyric acid (GABA) and alanine (Ala) in tea samples. Extracts from samples were diluted to eliminate matrix interference before introduction into the CE system. GABA and Ala in the diluted sample zone were derivatized with o-phthaldialdehyde/2-mercaptoethanol (OPA/2-ME) to form fluorescence-labeled products in the stacking process, and the labeled derivatives were then enriched by online stacking. Optimal conditions for the stacking, such as the concentration of the background buffer solution, the matrix of the sample zone (sample solution), and the volume of the sample injection, were investigated and then applied to real sample analysis. Under optimum conditions, the detections were linear in the range of 5.0 nM-2.5 microM with the square of correlation coefficients (R2) of 0.9995 and 0.9992 for GABA and Ala, respectively. Detection limits were found to be 0.7 and 0.8 nM for GABA and Ala, respectively. Tea samples were analyzed with recoveries between 92.33 and 97.87% and between 94.36 and 96.46% for GABA and Ala, respectively. This method is a rapid, convenient, and sensitive process for determining GABA and Ala in complicated matrix samples such as tea samples.