The preparation and characterization of a DNA biosensor based on kappa-carrageenan-polypyrrole-gold nanoparticles (KC-PPy-AuNPs) nano-biocomposite is reported to demonstrate an application for gender classification of Arowana fish. Immobilization of the thiol modified Arowana fish ssDNA probe sequence was successfully carried out via covalent attachment to the gold (Au) on the surface of the nano-biocomposite membrane. Under optimum conditions, the DNA biosensor showed a short immobilization and hybridization time with high sensitivity for monitoring the target DNA sequence. The hybridization with the target DNA was determined by monitoring the redox current of anthraquinone-2-sulfonic acid monohydrate sodium salt (AQMS) using differential pulse voltammetry (DPV). The DNA hybridization response gradually increased with increasing concentrations of target DNA from 5 × 10⁻¹⁸ to 5 × 10⁻¹² M and the limit of detection was below 5 × 10⁻¹⁸ M. The application of the DNA biosensor for gender classification of Arowana fish was demonstrated.