We have developed a novel optical DNA/protein sensor which consists of single-stranded (ss) DNA-Cy3 probes on gold surface and one line-shape polydimethylsiloxane (PDMS) microfluidic channel. This novel ssDNA-Cy3 probes, which does not require hairpin-like stem-loop conformation, show similar advantages as traditional molecular beacon technique in this microfluidic sensor. This chip-based optical sensor showed strong fluorescence enhancement by the binding of cDNA or cDNA-biotin. After introduction of cDNA-biotin, streptavidin can be determined by fluorescence quenching. This sensor showed strong affinity and high sensitivity toward streptavidin, the minimum detectable concentration for streptavidin was 1 pM, equating to an absolute detection limit of 50 amol in this sensor.