<p>Anaerobic digestion (AD) is essential for the global carbon cycle, playing a vital role in decomposing organic waste and generating renewable energy. Conventional single-phase AD systems often exhibit low efficiency and operational instability, whereas two-phase systems employing artificial separation of acidogenic and methanogenic phases offer a more efficient alternative for energy recovery, although at higher costs. This review provides a comprehensive analysis of two-phase AD processes and their operational efficacy, focusing on critical operational parameters: pH, temperature, substrate characteristics, hydraulic retention time, organic loading rate, and reactor configuration. The pivotal roles of feedstock pretreatment and inoculum adaptation in shaping structure and metabolic functions of microbial consortia are highlighted. These approaches may facilitate direct interspecific electron transfer mechanisms within the digestive system. Two-phase AD shows greater potential for development than single-phase AD due to its enhanced system stability and energy recovery efficiency. AD is widely recognized as the most cost-effective and practical method for organic waste treatment, energy recovery, and resource utilization. Nevertheless, two-phase AD implementation may face challenges related to technical feasibility, appropriate raw material selection, high operational costs and process innovation. This review offers novel insights into two-phase AD systems by examining process parameters at the macro level and microbial coordination mechanisms at the micro level.</p> Graphical abstract <p></p>