Biohydrogen production by fermentative bacteria is one of the most prospective alternative ways for fossil fuels. Therefore, this study aimed to isolate new facultative anerobic bacteria from cow rumen as highly hydrogen producers from sugarcane molasses. Among the isolated ruminant anerobic bacteria, the isolate RM92 was the highest hydrogen producer with maximum cumulative hydrogen (Hmax) of 426.67±14.56 mL/L. This bacterial isolate (RM92) was identified phenotypically and genotypically as Escherichia fergusonii. The 16S rRNA gene sequence was deposited in NCBI GenBank database under the accession number OP185369. Optimization expermients design for maximization of hydrogen production by E. fergusonii RM92 increased the Hmax to 1280.00 ±34.64 ml/L with maximum hydrogen production rate (Rmax) of 36.92±0.89 ml/L/h on 4% molasses sugar concentration, pH 8, incubation temperature 40 °C and initial inoculum size 30% (v/v). These findings suggest that E. fergusonii RM92 could be used as a potential hydrogen producer from cheap agro-industrial wastes.