The tumor microenvironment contains distinctive biomarkers, including acidic pH, elevated levels of reactive oxygen species (ROS), and hypoxia, necessitating the development of efficient biosensors for simplified cancer detection. This study presents an O₂-responsive hydrogel biosensor composed of [1,1′-biphenyl]-2,2′,4,4′,5,5′-hexaol (HDP) and polyvinyl alcohol (PVA) that exploits polyphenol-mediated interactions under N₂ and O₂ microenvironments. The oxidative susceptibility of the polyphenolic HDP moiety influences its distinct mechanical, physical, and electrochemical properties, allowing the differentiation between cancerous and normal cells. The in vitro assessments with cancer cell lines (HeLa and B16F10) and normal cell lines (CHO-K1) enabled distinctive electrical and mechanophysical outputs, as evidenced by enhanced mechanical compressive modulus and high conductivity, regulated by normoxic cellular states. In addition, the inherent ROS-scavenging capability of the HDP–PVA hydrogel sensor supports its potential application in hypoxia-related diseases, including cancer.