The unbridled release of harmful endocrine disruptors (EDs) into the environment is deteriorating human and animal health. A facile and efficacious biosensor was developed by immobilizing laccase over electropolymerized poly anthranilic acid on a carbon fiber paper (CFP) electrode, Lac/PAA/CFP for the detection of p-nonylphenol (PNP). PNP is a persistent phenolic endocrine disruptor and a harmful eco-toxic pollutant. Physico-chemical and electrochemical characterization of the fabricated electrode was carried out to study the modification of the Lac/PAA/CFP electrode. Cyclic voltammetric studies divulged that the prepared sensor has catalytic activity approximately twice greater than that of the bare CFP electrode. The influence of pH and scan rate was scrutinized for the modified electrode. Under optimized conditions differential pulse voltammetric studies were used for the quantification and the results revealed that the biosensor has a low limit of detection (LOD) and limit of quantification (LOQ) of 1.74 nM and 5 Nm, respectively with a broad linear dynamic range of 5–250 nM. In the presence of interferants, the developed biosensor exhibited good selectivity toward the electrochemical detection of PNP. Molecular docking studies carried out revealed the hydrogen bonding interaction of the Asn264 residue of laccase Trametes versicolor. Further, the fabricated biosensor was accessed for its practicality in real samples collected from tap water and lake water.