Lab-on-a-Chip (LOC) and μ-TAS (micro-total analytical system) are based on miniaturized integrated platforms that have the potential to revolutionize chemical, biological, and biochemical synthesis and analysis. Here, we demonstrated a process of fabricating a mosaic DNA chip and a corresponding detection method by time-resolved fluorescence (TRF) labeling. We synthesized oligonucleotide sequences in situ on glass slides directly, and then sliced them up into small pieces and patched up the pieces with different sequences to generate a mosaic DNA chip. With multiple BCPDA (BCPDA, abbreviated from 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid) labeling method based on biotin-avidin amplification, we established a TRF detection format on the mosaic DNA chip. The detection method allows discriminatory signals for perfect match, one-base mismatch, two-base mismatch and three-base mismatch by TRF labeled hybridization, whereby Europium (III, Eu3+) was captured and released on the principle of complexation and dissociation interaction between BCPDA and Eu3+ solution when the BCPDA-tagged avidin and biotin-ended oligonucleotide sequence linked. The fluorescence spectra and related lifetimes were determined. Also, we compared the TRF detection mode with the conventional fluorescence one. These results showed the former is more reliable and stable than the latter, especially for the mosaic DNA chip. Likewise, by applying TRF probing (or labeling) to specific bio-systems, the discovery is of fundamental interest and has significant implications to time-resolved-fluorescence based detection on biosensor.