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Molasses, Stillage or vinasse and Spent wash (bottom product of distillation column) are large liquid wastes production in the bioethanol industry that contain high sulfur. The liquid wastes with high sulfur contain are dangerous if it discharges directly into the environment without pretreatment. For this reason, pretreatment is needed to reduced the sulfur concentration of liquid waste (molasses) using biofiltration method. The objective of this research is to eliminate the content of H2S or sulfur in the wastewater of the bioethanol industry (molasses) by using aerobic bacteria such as Acidithiobacillus thiooxidans dan Pseudomonas putida. The method of this experimental work used biofiltration which are attached on wood chips or bagasse packing by aerobic bacterial to form biofilms in the reactor. The process in this study was conducted in two steps. The first step was carried out by sulfur oxidizing bacteria such as Acidithiobacillus thiooxidans and Pseudomonas putida with a concentration of 10% and 20% (v/v) that growth on packing to form biofilms in the reactor for 13 days. Furthermore, as the second step the bacteria degraded H2S content of liquid waste with attached bacteria on packing for 15 days in biofiltration reactor. From the preliminary results of this study, reactor with 10% (v/v) A. thiooxidans on wood chips packing and 3% (v/v) concentration molasses waste can degraded H2S from 4.35 mg/L to 1.55 mg/L (64.37% removal efficiency) and for reactor with 20% (v/v) A. thiooxidans can degraded H2S from 4.35 mg/L to 1.16 mg/L (73.33% removal efficiency). Meanwhile, reactor with 10% (v/v) Pseudomonas putida can degraded H2S from 4.35 mg/L to 1.94 mg/L (55.40% removal efficiency) and for reactor with 20% (v/v) Pseudomonas putida can degraded H2S from 4.35 mg/L to 1.47 mg/L (66.21% removal efficiency).