The polymer waveguide optical biosensor based on the Mach-Zehnder interferometer (MZI) by using spectral splitting effect is investigated. The MZI based biosensor has two unequal width sensing arms. With the different mode dispersion responses of the two-arm waveguides to the cladding refractive index change, the spectral splitting effect of the output interference spectrum is obtained, inducing a very high sensitivity. The influence of the different mode dispersions between the two-arm waveguides on the spectral splitting characteristic is analyzed. By choosing different lengths of the two unequal width sensing arms, the initial dip wavelength of the interference spectrum and the spectral splitting range can be controlled flexibly. The polymer waveguide optical biosensor is designed, and its sensing property is analyzed. The results show that the sensitivity of the polymer waveguide optical biosensor by using spectral splitting effect is as high as 104 nm/RIU, with an improvement of 2–3 orders of magnitude compared with the slot waveguide based microring biosensor.