We present a sensitive, simple and robust on-chip transient ITP (TITP) method for the analysis of polymerase chain reaction (PCR) samples. This TITP analysis was performed on a poly (methyl methacrylate) microchip with cross structure. The PCR sample was injected into a 4 mm free-solution buffer channel to increase sample loading. When applied separation voltage, the sample ions were stacked by TITP using chloride ions in the sample matrix as the leading ions and HEPES in the background electrolyte as the terminating ions. The stacked sample plug was then separated by zone electrophoresis (ZE) in hydroxypropylmethyl cellulose polymer. The whole operation was carried out in single background electrolyte. Sample injection, preconcentration / separation were performed continuously with the sequential switching of voltage. By injection of the sample into the free-solution buffer channel, sampling bias was avoided and the injection time was reduced. With this TITP method, the detection sensitivity was improved without loss of resolution, a 20-fold signal enhancement was achieved and the average limit of detection was estimated to be 0.24 pg/mu L (S/N = 3). The TITP method is simple, fast and sensitive, thus is well suited to direct analysis of the highly saline PCR samples.