Amphiphilic lipids can often form various lipid-based lyotropic liquid crystalline phases when they are exposed to aqueous environments, which includes the inverse hexagonal (H2), inverse cubic (V2), inverse discontinuous cubic (I2) and lamellar (Lα) phases. Their different interactions with crossed-polarised light make some phases appear bright and some dark, which offers great potential in developing a novel universal assay platform using birefringence as the visual signal output. Here, we have developed a novel strategy for constructing an assay platform using lyotropic liquid crystalline phases as the signal transducer and using changes in their birefringence upon exposure to lipase as the signal output. The hydrolysis of the ester group of glyceryl monooleate (GMO) by lipase induced a phase transition from cubic phase to hexagonal phase and consequently "turned on" the birefringence as the signal output. The change in the intensity of the birefringence depended on both the exposure time and concentration of lipase. The streptavidin-biotin affinity was utilised to demonstrate the potential of the birefringence assay platform, using free biotin as the model analyte, biotinylated lipase and streptavidin-coated magnetic beads in the competitive format. A semi-quantitative assay with a detection limit of 5 μg mL-1 to free biotin as the model analyte was achieved in the visual birefringence mode. This study demonstrated a proof-of-concept lyotropic assay platform with birefringence as the visual signal output that could be deployed as an electronics- and colour-free diagnostic device for a wide range of applications, for example to indicate the presence of toxins in water.