A novel sandwich assay with molecular beacons as report probes has been developed and integrated into one-dimensional microfluidic beads array (1-D chip) to pursue a label-free and elution-free detection of DNA/mRNA targets. In contrast with the immobilized molecular beacons, this sandwich assay can offer lower fluorescence background and correspondingly higher sensitivity. Furthermore, this sandwich assay on 1-D chip operating in conjunction with molecular beacon technique allows multiple targets detection without the need of laborious and time-consuming elution, which makes the experiment process simple, easy to handle, and reproducible results. In the experiment, the synthesized DNA targets with different concentrations were detected with a detection limit of approximately 0.05 nM. Moreover, the mRNA expression changes in A549 cells before and after anticancer drug 5-flouorouracil treatments were detected and the results were validated by the conventional RT-PCR method.