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A low-cost, small-size, and all-solid-state biosensor for the specific determination of L-ascorbic acid was developed. The sensor array was fabricated by screen printing, and the ruthenium oxide (RUO2) thin film was deposited on the working area of each sensor by an RF sputtering method for surface modification. A conductive polymer (polypyrrole) miniaturized reference electrode was used in combination with the biosensor. The ascorbate oxidase was immobilized with 3-glycidoxypropyltrimethoxysilane (GPTS) methods in the bioactive membrane layer. In accordance with experiment results, the sensor has the better average sensitivity of 51.85 mV/pH between pH 1 and pH 13. The investigated characteristics in the polypyrrole film modified reference electrode show a very small drift voltage (5 mV/h) and the standard deviation of the reference electrode is 1.093 mV. According to the experiment results, the potential response of the enzyme-based biosensor depends linearly on L-ascorbic acid concentration between 0.02 mM and 1 mM with a linear correlation coefficient of 0.86 and the sensitivity is 13.85 mV/mM.