Phenylketonuria (PKU) is a congenital disease. The mutations in a specific gene that expressed enzyme phenylalanine hydroxylase, lead to the high level of amino acid phenylalanine (phe) and PKU disease [1]. PKU screening is done after brith because the higher amount of phe caused to brain damage and mental retardation [2].Therefore, phe determination is important in biological samples and analytical method such as chromatography has been applied for phe analysis. Present methods are expensive and time-consuming and development of the fast, cheap, easy and portable techniques are noteworthy. In this research, a novel and facile method for determination of phe by immobilization of phenylalanine hydroxylase in the mosses leaf-like extract at fiber matrices of the modified paper disc was developed. Immobilization of the plants tissue as the source of enzymes has been applying recently [3]. We used the added potassium hexacyanoferrate to the enzymatic paper disc as a mediator. For modification and increasing sensitivity, ZnO@Au nanohybrid was added within the paper disc. This paper disc was placed on the top of the graphite screen printed electrode and was connected to potentiostat/ galvanostat electrochemical device. Characterization of synthesized nanostructure was down by scanning electron microscopy and Fourier transform infrared spectroscopy. The electrochemical behaviour of this biosensor was studied by cyclic voltammetry and electrochemical impedance spectroscopy methods. After optimization of effective parameters, phe measurements were done by differential pulse voltammetry. This biosensor showed a linear response for phe concentrations with an appropriate detection limit. The stability of this biosensor and its selectivity against some interferences biomolecules were evaluated. The practical applicability of the biosensor was established for the voltammetric determination of phe in the heparinized blood plasma sample and statistical tests showed usability of the proposed biosensor.