In this study a simple and selective methodology was used to develop a novel electrochemical DNA biosensor for determination of flutamide. This biosensor was made of pretreated screen printed electrode(SPCE) that modified with reduced graphene oxide(RGO) decorated with ds-DNA, This simple and inexpensive biosensor was performed through the interaction of small molecules such as flutamide with the DNA. First, the intraction of salmon sperm ds-DNA with flutamide on the surface of ds-DNA modified screen printed electrode was studied. The oxidation signals of guanine and adenine were used as probs to study the intraction. By intraction of flutamide with the DNA, the oxidation currents of guanine and adenine were decresed at the modified-SPCE, using differential pulse voltammetry. A linear dependence of the adenine oxidation signal was observed for the determination of flutamide concentration in the range of 0.0025- 0.075 and 0.075- 7.5 nmol L -1 (at the optimum conditions) with a detection limit of 0.0015 nmol L -1 for the adenine signal. The relative standard deviation for four replicate measurements of 0.05 nmol L -1 flutamide was 4.5%, for adenine. This method is successfully was applied for determination of a few amount of flutamide in real samples.