The present study aims to enhance the efficiency of anaerobic sludge microorganisms to produce hydrogen (H₂) through various pre-treatment methods. The various pre-treatment methods such as base, acid, chloroform, heat shock, freezing and thawing have enabled to isolate acidogenic bacteria with higher bio H₂ producing activity in an anoxic environment. From these various treatments, bacteria were isolated and screened for bio H₂ capabilities. Among the one bacterial strain, AH4 strain showed maximum cumulative H₂ production and Hydrogen Yield (HY) using 100% diary anaerobic sludge. AH4 strain was identified as Acinetobacter junii using 16S rRNA gene sequence and used for further experimental analysis. Biohydrogen productions of Acinetobacter junii were measured at different experimental setup such as various pH levels (5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) and different substrate concentration (10 – 100%) of dairy anaerobic sludge substrate. At pH 7.5 and 60% substrate concentration, the strain AH4 Acinetobacter junii displayed the maximum cumulative H₂ production of 945.7 ml/L and H₂ yield 1.35 mol H₂/mol glucose. Based on our results, we concluded that Acinetobacter junii can be used as a promising bio agent for hydrogen production on a large scale using dairy anaerobic sludge as substrate.