We present a simple platform of poly-eriochrome Black T decorated with nickel hydroxide nanoparticles (Ni(OH)2NPs/PEBT) for the electrocatalytic oxidation of streptomycin (STRP) and probing its interaction with double-stranded DNA (ds-DNA). The electropolymerized eriochrome Black T modified glassy carbon electrode (GCE) was used to stabilize and synthesize Ni(OH)2NPs. The oxidation of STRP on Ni(OH)2NPs/PEBT/GCE mainly proceeded as a surface mechanism with the contribution of a diffusion-controlled process. A significant catalytic oxidation current of STRP was measured from an alkaline medium. It showed a dependence on the STRP concentrations in the range of 0.1 to 1.0 mM with a detection limit of 0.04 mM. The sensor has shown good recovery (85.3 % to 90.2 %, with RSD range of 1.27 to 8.83 %) for the detection of SPRP in diluted human serum. The interaction of STRP with ds-DNA was investigated from phosphate buffer pH 7.4 (0.2 M) using different electrode modifications (PEBT/GCE, and Ni(OH)2NPs/PEBT) in addition to bare GCE using voltammetric and impedance techniques. The binding constant of the STRP-DNA complex was calculated. Under the studied conditions, the interaction of ds-DNA with STRP in solution or on the surface of PEBT/GCE demonstrates their binding in intercalative and electrostatic modes.