The paper proposes an efficient approach applied in DNA base calling, which concerns efficiency and sensitivity. We utilized the Neuro-Fuzzy model in the analysis issues to determine the confidence value prediction in DNA base calling, that is solved by several attempts applied in the MATLAB tool, the model is implemented for the collected data for each base in the DNA sequencing. The model is designed by using the ANFIS tool, which contains three subsystems a main system. We obtain three features (peakness, height, and spacing) for each base from the three subsystems and in the main system use these three features as the input to predict the confidence value for each base in the DNA. This achieves a high accuracy in the obtained results with high-performance.