We present an aptamer-based biosensor (aptasensor) for rapid and high-sensitive detection of oxytetracycline (OTC) antibiotic in PBS inside a Y-channel PDMS microfluidic device. The detection was made by real-time monitoring of the agglutination assay of ssDNA aptamer-conjugated polystyrene latex microspheres with proximity optical fibers. The agglutination assay was performed with serially diluted OTC antibiotic solutions using highly carboxylated polystyrene particles of 920 nm diameter conjugated with OTC-binding ssDNA aptamer. Proximity optical fibers were used to measure the increase in 45° forward light scattering of the aggregated particles by fixing them around the viewing cell of the device with stable angle and distance to the detector. The detection limit was around 100 ppb for the current aptasensor system with the detection time less than 3 min.