A new conceptual technique for a fast DNA hybridization detection is developed.Here, we report a fast and sensitive on-line fluorescence resonance energy transfer (FRET) detection technique of label-free target DNA, mouse gene F9.The method is based on changes of the FRET signal caused by the sequence-specific hybridization between two fluorescently labeled nucleic acid probes and target DNA in a pillar-type microfluidic channel.Confocal laser-induced microscope has been used for the detection of fluorescence signal changes.In the present study, target DNA sequences were not amplified using PCR since the sensitivity of confocal laserinduced fluorescence detection is very high.Three DNA oligonucleotides (TET-probe: 5-CTGA TTAG AGAG AGAA-TAMRA-3; TAMRA-probe: 5-TET-ATGT CTGA GCTG CAGG-3 and Target DNA: 3-GACT AATC TCTC TCTTA CAGG CACT ACAG ACTC GACG TCC-5) are simultaneously introduced into the channel by a microsyringe pump, and they are efficiently mixed by passing through the micro-pillar type chennel.Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to a real-time analysis of DNA targets in a solution phase.