A two dimensional photonic crystal biosensor with high quality factor, transmission and sensitivity has been theoretically investigated using two dimensional finite difference time domain method (FDTD) and plane-wave expansion (PWE) approach. The studied biosensor consisted of two waveguide couplers and one microcavity formed by removing one air pore. Following analyte injection into the sensing holes and binding, the refractive index changes inducing a possible shift in the resonant wavelength. For the optimized structure, the biosensor quality factor is found to be over 49,767 and the obtained sensitivity is of order 15.2 nm/fg. Also, we investigated this structure in case of all air holes are applied as the functionalized holes with a sensitivity was found to be approximately equal to 292.46 nm∕RIU (refractive index units). According to the resonance cavity characteristics, the demultiplexing of different wavelengths can be achieved by regulating the radius of defects “r” inside the cavity. For this, A new design with 2D PCs for two-channel demultiplexer optofluidic biosensor has been proposed. The analysis shows that the response of each channel has a different resonant cavity wavelength and the filling of analyte in the selected holes cause resonant wavelength shifting, independently.