We reveal a novel design for dye-doped liquid crystal (DDLC) microfluidic biosensing chips in the polydimethylsiloxane material. With this design chip, the orientation of DDLCs was affected by the interface between the walls of the channels and DDLCs. When the inside of a channel was coated with an N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP) alignment layer, the DDLCs oriented homeotropically in a homeotropic (H) state under cross-polarized microscopy. After immobilization of antigens with antibodies on the alignment layer-coated microchannel walls, the optical intensity of the DDLC change from the dark H state to the bright planar (P) state. Using pressure-driven flow, the binding of antigens/antibodies to the DDLCs could be detected in an experimental sequential order. The microfluidic DDLCs were tested by detecting bovine serum albumin (BSA) and its immune-responses of antigens/antibodies. We proved that this immunoassay chip was able to detect BSA antigens/antibodies pairs with the detection limit about 0.5 µg/mL. The novel DDLC chip was shown to be a simple, multi-detection device, and label-free microfluidic chips are presented.