Bio-electrochemical systems have increasingly become the focus of research due to their potential in environmental biotechnology, particularly in the domains of waste utilization and energy recovery. A prominent method within this domain is the transformation of organic matter into hydrogen via microbial electrolysis cells (MECs). This study offers a thorough analysis of MEC performance, employing exergy analysis and incorporating relevant data from the existing literature. The findings of this research indicate a relationship between process efficiency and effective electron transfer originating from biological oxidation to the cathode reaction, facilitating hydrogen generation. The assessment performed revealed that the exergy efficiency of the process varies by a wide range, depending on conditions such as substrate type and concentration, applied external voltage, and the presence of specific inhibitors. This interplay between substrate concentration, overall efficiency, and energy requirement underlines the complex dynamics of optimizing MEC performance. Our insights provide understanding of the challenges in bio-electrochemical systems, offering implications for their sustainable and efficient use in environmental biotechnology. The theoretical analysis involved assessing the utilization of glucose and glycerol, along with the evaluation of electrical energy consumption and hydrogen yield. Our results demonstrate that a higher applied voltage is associated with greater exergy efficiency. Furthermore, after comparing the use of glucose and glycerol as substrates, our study supports the preferential application of glucose for enhanced efficiency.