A high-sensitivity, high-resolution, multi-working channel and real-time detection typed one-dimensional photonic crystal biosensor is proposed. Through changing the slit’s width between two adjacent silicon layers,multiple defects with different resonant wavelengths can be designed. The biosensor is based on the air-mode cavity which means light energy is greatly limited to the defect channels. Owing to the strong localizing capability of the defects and the forceful light-material interaction, a subtle alteration of the liquid’s refractive index can make the resonant wavelength and transmittance changes significantly. Due to the mutual coupling effect of multiple defect modes, when the liquid’s refractive index around a defect changes, the resonant wavelengths of all the device’s defect modes and the corresponding transmittances will be affected. Different liquid samples can flow freely within respective slits, so they can be measured in real time and at the same time. Through analyzing the alterations of the wavelengths and transmittances, the concentration information of a variety of liquid samples can be measured through a wide transmission spectrum measurement.