A novel chemosensor has been developed for the accurate and sensitive detection of Hg²⁺ ions in industrial wastewater. This sensor uses a stick-like nanocellulose architecture synthesized via a green method. The unique morphology and surface area of nanocellulose make it an ideal mesoporous substrate for immobilizing the chromophore 1-(benzothiophenyl)-3-(benzooxazolyl)-2-((4-bromophenyl)diazenyl)propane-1,3-dione (azo-dione ligand, ADOL). Comprehensive characterization of the fabricated chemosensor and its nanocellulose base was carried out using FTIR, SEM, TEM, BET surface area, and XRD to evaluate their structural and morphological properties. Spectrophotometric parameters, including pH, response time, selectivity, and sensitivity, were extensively optimized to ensure optimal sensing performance, enabling detection of Hg²⁺ at very low concentrations. Method validation was performed in accordance with ICH (International Council for Harmonisation) guidelines, confirming the reliability of the sensor in terms of limit of detection (LOD), limit of quantification (LOQ), linearity, and precision. The spectrophotometric method achieved a highly sensitive LOD of 9.07 µg L⁻¹. Moreover, the ADOL chemosensor demonstrated excellent reusability, maintaining performance over five cycles following regeneration with 0.1 M thiourea, underscoring its sustainability. Finally, the sensor exhibited outstanding performance in detecting Hg²⁺ across various industrial wastewater samples, highlighting its practical applicability, exceptional selectivity, and high sensitivity for real-world environmental monitoring.