Background <p>A cholesteric liquid crystal (CLC) system comprising the calamitic LC 8CB (4-octyl-4’-cyanobiphenyl) doped with the chiral agent R5011 was applied as a biosensing medium.</p> Results <p>By exploiting the pronounced light-scattering behavior at the smectic-to-chiral nematic transition temperature of 26.5&#xa0;°C of the vertically aligned CLC, a distinct red–green (signal–background) color contrast was observed in the optical texture of the CLC in the presence of the protein standard bovine serum albumin (BSA) or the cancer biomarker CA125, with the red color intensity correlated positively with the concentration of the biological analyte. The high-contrast signal–background color scheme of the 8CB/R5011 CLC was confirmed through theoretical calculations to simulate the interference spectra and the corresponding color observed under a polarizing optical microscope. The simulation results also indicate that the collective tilt angle of the CLC increased in the presence of biomolecules, suggesting that the alignment of CLC was disrupted and light scattering was enhanced. By incorporating haze measurements as the quantitative approach for the CLC-based biosensor, a strong correlation between the mean haze value and the analyte concentration was demonstrated. The limit of detection (LOD) achieved through haze analysis was 1.23 × 10⁻¹² g·mL⁻¹ for BSA and 3.85 × 10⁻¹⁰ g·mL⁻¹ for CA125, which were substantially lower than those obtained via conventional image analysis (2.21 × 10⁻¹¹-g·mL⁻¹ BSA and 1.43 × 10⁻⁹-g·mL⁻¹ CA125). In a proof-of-concept demonstration with CA125-spiked human serum as the analyte, the LOD remained unaffected by interferents present in human serum.</p> Conclusions <p>The CLC-based biosensing technology combined with haze-based quantitation offers a label-free, highly sensitive, rapid response, cost-effective, and versatile platform for detecting biological analytes, manifesting significant potential for applications in early disease diagnosis and biomedical research.</p>