Biomass of Lactobacillus acidophilus was optimized in the tofu wastewater medium by Plackett-Burman matrix and Response surface methodology – Central composite design (RSM-CCD). The highest biomass was 0.73 g/100mL at 106 CFU/mL of initial density of L. acidophilus in tofu wastewater medium, with 0.37% (NH4)2SO4 and 17.59% sucrose. L. acidophilus produced maximum value of biomass after 20 hours of incubation at 37°C. There was a similar proportion at 96.05% in comparison to experimental value and optimization theory, approximatly 93.58% L. acidophilus biomass in MRS medium. As a result, optimization model could be applied in biomass production of L. acidophilus to enhance comercial value and contribute to environmental protection.