We report concentration methods for the analysis of small solutes by capillary electrophoresis in conjunction with laser-induced native fluorescence using a Nd:YAG laser. After injecting samples, poly(ethylene oxide) (PEO) in the anode reservoir entered a capillary filled with Tris-borate buffer. When migrating in PEO solution, the analytes slowed down and stacked at the interface between the sample zone and PEO solution. As a result, the limits of detection (LODs) down to 8 pM for 2-naphthalenesulfonic acid and 70 pm for L-tryptophan have been achieved when injecting at 30 cm height for 120 s and 230 s, respectively. Such low LODs are partially due to the effects of NaCl in the samples and PEO on the fluorescence characteristics of the analytes. In addition, the concentrations of NaCl and PEO have great impacts on the migration of the analytes and electroosmotic flow, thereby affecting resolution and speed. Without pretreatment, the determinations of five important markers in urine samples and two acids in a cerebrospinal fluid sample have been performed separately, with the relative standard deviations of the concentrations less than 3.6%. Furthermore, by applying a short plug of low-pH buffer after injection, the analysis of greater volumes of the urine sample has been carried out, resulting in detecting more peaks.