The direct discharge of cationic surfactants into environmental matrices has exponentially increased due to their wide application in many products. These compounds and their degraded products disrupt microbial dynamics, hinder plant survival, and affect human health. Therefore, there is an urgent need to develop electroanalytical assessment techniques for their identification, determination, and monitoring. In our study, ZnO-PANI nanocomposites were electrodeposited on a glassy carbon electrode (GCE), followed by the immobilization of laccase enzymes and the electrodeposition of polypyrrole (PPy), to form a biosensor that was used for the detection of CTAB. A UV-Vis analysis showed bands corresponding to the π-π* transition of benzenoid and quinoid rings, π-polaron band transition and n-π*polaronic transitions associated with the extended coil chain conformation of PANI, and the presence and interaction of ZnO with PANI and type 3 copper in the laccase enzymes. The FTIR analysis exhibited peaks corresponding to N-H and C-N stretches and bends for amine, C=C stretches for conjugated alkenes, and a C-H bend for aromatic compounds. A high-resolution scanning electron microscopy (HRSEM) analysis proved that PANI and ZnO-PANI were deposited as fibres with hairy topography resulting from covalent bonding with the laccase enzymes. The modified electrode (PPy-6/GCE) was used as a platform for the detection of CTAB with three linear ranges of 0.5–100 µM, 200–500 µM, and 700–1900 µM. The sensor displayed a high sensitivity of 0.935 μA μM−1 cm−2, a detection limit of 0.0116 µM, and acceptable recoveries of 95.02% and 87.84% for tap water and wastewater, respectively.