Ochratoxin A (OTA) as the most dangerous mycotoxin is produced by Aspergillus Ochraceus and Penicillium verrucosum. OTA can be found in beverages and foodstuffs that induces the teratogenic, nephrotoxic, carcinogenic, and immunosuppressive effects on humans. Hence, developing highly sensitive methods for its detection is of great importance. Herein, a novel aptasensor was designed for the label-free monitoring of the ultra-low OTA levels by a combination of the superiority of aptamers and long-range orientational order of liquid crystals (LCs). The aptasensing strategy was based on the conformational switch of the immobilized π-shaped DNA structure on the glass substrate in presence of the target. A shift in the orientation of LCs from random to homeotropic state led to the apparent alteration of the optical appearance of the aptasensor platform from bright to dark. The LC-based aptasensor especially detects OTA at the ultra-trace level as low as 0.63 aM with comparable selectivity. The aptasensor could detect OTA successfully in the grape juice, coffee, and human serum samples. The LC-based aptasensor paves a way for developing portable and real-time sensing probes with high performance for food safety control and clinical application.