Assessment of biogas production from Napier Grass (NG) in co-digestion with cattle dung (CD) was carried out in laboratory scale reactors and optimization using response surface methodology (RSM) and Box-Behnken design of the experiment. The effects of total alkalinity (TA), volatile solids (VS), pH, and volatile fatty acids (VFA) at three levels were investigated along with gas production. In this study, we determined the optimal ratio for biogas generation from NG and CD co-digestion. The three blending ratios were adopted as NG:CD (50:50), NG:CD (65:35), and NG:CD (75:25). The optimized result revealed that the highest generation of biogas was achieved at the blending ratio NG: CD (65:35) up to 0.4813 m3/kg VS. However, the significant value of R2 (0.9825) during RSM optimization highlighted that, the model might be effectively used to forecast the generation of biogas from the blending of CD and NG. The result shows that TA, VS, pH and VFA are essential for biogas production and the model algorithm could be applied extensively to estimate biogas generation from the co-blending of various organic biomasses. There is a good correlation between each parameter and the overall generation of biogas in the ANOVA results.