Liquid crystals (LCs) are soft matters exhibiting both the fluidity of liquids as well as the ordered molecular orientation of crystals. They are optically birefringent and are responsive to various external stimuli such as heat, light, electric and magnetic fields. LC-based biosensors are one of the emergent fields in the biomedical application of LCs. Conventional LC-based biodetection at the LC‒glass interface requires the assembly of a LC cell using a pair of glass substrates. To simplify the sensing platform, a single glass substrate spin-coated with a thin film of LC was used for the detection of bovine serum albumin and the cancer biomarker CA125. The single-substrate biosensor achieved a similar or lower limit of detection compared to that based on a LC cell. We also explored the biosensing application of lyotropic LCs, a hydrophilic and therefore more biocompatible counterpart of thermotropic LCs, which are the major sensing media of current LC-based biosensing techniques. Our results demonstrate that the nematic phase of lyotropic chromonic LCs (LCLCs) can be employed in quantitative optical biosensing with the potential capability of real-time detection.