Here we have developed a self-referencing (SR) biosensing platform where several dye-doped polystyrene microspheres (of approximately equal size) are integrated into the microfluidic channel of a microfluidic chip. To demonstrate specific interaction with different analytes, the surface of some microspheres is functionalized, while other microspheres acts as dynamic reference to exclude the signal fluctuations caused by environmental changes or nonspecific binding events. By monitoring the resonance wavelength separation between the sensing spheres and the reference spheres of the SR-biosensing device, we have realized the detection of bovine serum albumin. This SR-biosensing device can be used in biomedical diagnostics due to demand for real time noninvasive techniques.