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The development of highly efficient and stable electrocatalysts for their application in electrochemical detection is desirable. Herein, a new copper nanowires (CuNWs) modified electrode was fabricated by coating poly(dimethyl diallyl ammonium chloride) (PDDA) stabilized CuNWs composite onto the the glassy carbon electrode (GCE), and used for electro-catalyze and determination of cysteine (CySH). The morphology, structure, and surface property of fabricated CuNWs were tested using SEM, XRD, and XPS, separately. Electrocatalytic behavior of the modified electrode (CuNWs-PDDA/GCE) for CySH oxidation in alkaline medium was researched by cyclic voltammetry (CV) and chronoamperometry (CA) thoroughly, displaying excellent electrocatalytic oxidation activity and sensing ability towards CySH with a rapid and sensitive anodic current response at low oxidation potential. In the amperometric detection of CySH, the as-prepared electrode displayed a significantly wide linear range from 2 μM to 260 μM and a detection limit of 0.19 μM with a sensitivity of 1.430 μAμM−1cm−2. The proposed CySH sensor exhibited good reproducibility, stability, and anti-interference ability, and has been used to detect CySH in human plasma samples successfully with good applicability.