The properties of poly (2-methoxyaniline-5-sulfonic acid) (PMAS), a water-soluble polymer, has been investigated as a potential mediator for biosensors. Addition of poly (L-Lysine) (PLL) to the PMAS renders this material water-insoluble; an important feature for aqueous based biosensors. Characterisation of various PMAS/PLL mixtures showed that a uniform and stable film could reproducibly be fabricated on ITO coated Mylar using 0.1% (w/v) PMAS and 0.05% (w/v) PLL. Immobilisation of enzyme horseradish peroxidase (HRP) onto the PMAS/PLL film was also investigated. Amperometric detection (-0.1 V vs. Ag/AgCl reference electrode) of hydrogen peroxide was investigated using the PMAS/PLL/HRP electrode as the working electrode in a three electrode cell containing phosphate buffer saline (pH 6.4) solution. Amperometric responses were observed upon the addition of hydrogen peroxide. HRP immobilised concentration was optimised to be 0.35% (w/v) at 35 μL. Loading volume was also studied and optimised to be 35 μL. The optimal condition for amperometric test was using constant applied potential at -100 mV. Detection limit of the sensor in PBS solution was 0.01 mM H[2]O[2]. Calibration curve in PBS pH 6.4 shows a linear response range between 0.01 and 0.1 mM H[2]O[2] with a sensitivity of 24.91 μA/cm[2] mM[-1] and correlation coefficient of 0.9966.