Response Surface Methodology (RSM) based on Central Composite Design (CCD) was employed to optimize the conditions of enzymatic hydrolysis and fermentation of the seaweed Eucheuma cottonii. The significant influence of cellulose loading, enzyme loading and incubation time in enzymatic hydrolysis that has been screened by Plackett-Burman Design (PBD) was optimized using CCD. The optimum glucose concentration of 24.24 g/L (81% glucose yield) was obtained at 3% (v/v) of cellulose loading, 4% (v/v) of enzyme loading and 54 h incubation time. Subsequently, PBD analysis showed the significant effects of inoculum concentration, pH, temperature and time on fermentation process. Further optimization study by CCD revealed that 12% (v/v) of inoculum concentration, pH 5.2, 32 °C and 72 h of fermentation time enhanced the bioethanol production up to 9.77 g/L with the yield of 0.40 g/g and 78% conversion efficiency. Thus, the RSM based optimization of bioethanol from Eucheuma cottonii showed satisfactory results in this research. In short, it can be concluded that this optimization approach will serve as a good foundation for the realization of a consistent bioethanol production in the future. •Eucheuma cottonii seaweed was employed as the main feedstock for bioethanol production.•Enzymatic hydrolysis and fermentation were the main conversion technologies utilized.•Response Surface Methodology was used to optimize the enzymatic hydrolysis and fermentation for bioethanol production.