This paper presents a polydimethyl-siloxane (PDMS) microlens bonding on a compact disk (CD) microfluidic device that performs an enzyme-linked immunosorbent assay (ELISA) to enhance the sensitivity of bio-detection. The PDMS microlens can be fabricated with ease using soft replica molding method. A commercial software tool (ZEMAX) has been established to analyze the focal length of the microlens. A fluorescence detection system comprising of a PDMS microlens, a CD microfluidic device and an optical detection module is demonstrated. Consequently, using the developed microlens, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microfluidic devices were significantly improved. Successful detected the fluorescent-labeled beads is demonstrated using the developed method.