The present research describes a simple method for the identification of the gender of arowana fish (Scleropages formosus). The DNA biosensor was able to detect specific DNA sequence at extremely low level down to atto M regimes. An electrochemical DNA biosensor based on acrylic microsphere-gold nanoparticle (AcMP-AuNP) hybrid composite was fabricated. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesised with a facile and well-established one-step photopolymerization procedure and physically adsorbed on the AuNPs at the surface of a carbon screen printed electrode (SPE). The DNA biosensor was constructed simply by grafting an aminated DNA probe on the succinimide functionalised AcMPs via a strong covalent attachment. DNA hybridisation response was determined by differential pulse voltammetry (DPV) technique using anthraquinone monosulphonic acid redox probe as an electroactive oligonucleotide label (Table 1). A low detection limit at 1.0 × 10⁻¹⁸ M with a wide linear calibration range of 1.0 × 10⁻¹⁸ to 1.0 × 10⁻⁸ M (R ² = 0.99) can be achieved by the proposed DNA biosensor under optimal conditions. Electrochemical detection of arowana DNA can be completed within 1 hour. Due to its small size and light weight, the developed DNA biosensor holds high promise for the development of functional kit for fish culture usage.