In this review, selected examples are presented to demonstrate how microfluidic approaches can be utilized for investigating microbial life from deep geological environments, both from practical and fundamental perspectives. Beginning with the definition of the deep underground biosphere and the conventional experimental techniques employed for these studies, the use of microfluidic systems for accessing critical parameters of deep life in geological environments at the microscale is subsequently addressed (high pressure, high temperature, low volume). Microfluidics can simulate a range of environmental conditions on a chip, enabling rapid and comprehensive studies of microbial behavior and interactions in subsurface ecosystems, such as simulations of porous systems, interactions among microbes/microbes/minerals, and gradient cultivation. Transparent microreactors allow real-time, noninvasive analysis of microbial activities (microscopy, Raman spectroscopy, FTIR microspectroscopy, etc.), providing detailed insights into biogeochemical processes and facilitating pore-scale analysis. Finally, the current challenges and opportunities to expand the use of microfluidic methodologies for studying and monitoring the deep biosphere in real time under deep underground conditions are discussed.