Liquid crystals (LCs) can be employed in biological sensing and applied to label-free immunodetection owing to their unique birefringent, anchoring, alignment and collective properties. Like all different kinds of immunoassays, both sensitivity and specificity are universally the most important key points of concern. In this study, we developed various approaches toward ultrahigh sensitivity in LC-based immunoassays for potential clinical applications. The LC-based immunodetection technique was demonstrated with the cancer biomarker CA125, which is a mucin-like glycoprotein commonly present in the serum of patients with ovarian and other types of cancer. By using LCs with larger birefringence, such as HDN, the sensitivity of immunodetection was drastically enhanced compared to 5CB, which has a relatively lower birefringence and is commonly used in LC biosensing studies. In addition, UV modification of the monolayer of dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAP), which functions as the alignment layer for LCs, is suggested to increase functional groups suitable for covalent binding of biomolecules, stabilizing the immobilized anti-CA125 antibody and the immunocomplex thus formed, and contributing to the lowered detection limit. Finally, we show that it is possible to directly identify the formation of CA125 immunocomplex with HDN in a mixture of antigen and antibody without the need to eliminate unbound or unreacted biomolecules through washing, thereby creating a simplified procedure for faster LC-based immunoassay. It is evident from our results that label-free immunodetection based on birefringent LCs represents a novel biosensing technique with potentials to detect a wide range of biomolecules, providing an alternative to conventional label-based immunoassays.