Considering the ongoing climate change and increasing energy demand, hydrogen (H2) as a fuel represents a great capacity to serve potential form of renewable energy. Hydrogen can be synthesis using various processes, but the biological method for producing H2 had been strongly emerged during the previous few decades. Biohydrogen production from renewable sources through biological or photobiological processes had gained remarkable global attention from scientists or experimenter’s due to its promising role as an environmentally friendly fuel. For enhancing the biohydrogen production from microorganisms, advancements in bioengineering at both molecular and metabolic levels are significantly important. A wide range of naturally engineered microbes are found in nature exhibiting unique metabolic specialization. These microbes use different chemical compounds or substrates (as electron acceptors) to facilitate their growth and liberating electron as hydrogen donor. These fundamental outcomes, which have important industrial applications, lead to the use of metabolic engineering techniques aimed at improving biological hydrogen production from microbial communities. This enhancement is achieved through strategies such as the use of dark fermentation technique, over-expression of hydrogen producing enzymes, finding different metabolic routes, alleviating bottlenecks in metabolic pathways, and the deletion of non-functional genes. These methods significantly depend on choosing a right strain of microbes to produce biohydrogen. Currently, various molecular methods, for example, terminal restriction fragment length polymorphism (TRFLP), quantitative real-time polymerase chain reaction (q-PCR), fluorescent in-situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), ribosomal intergenic spacer analysis (RISA), next -generation sequencing (NGS), etc. had been used for enhancing the biohydrogen production from microbial communities. This book chapter describes the development of biohydrogen as a commercially feasible fuel through the fundamentals of molecular techniques and applications of metabolic engineering approach for improving the yield and quality of biohydrogen.