Abstract: Microalgae and cyanobacteria are regarded as the most promising third-generation biofuel feedstocks due to their advantages of non-arable land occupation, rapid growth rates, and strong CO₂ fixation capacity. However, profound differences exist between eukaryotic microalgae and prokaryotic cyanobacteria in terms of hydrogenase oxygen tolerance, cell wall structure governing anaerobic digestion kinetics, and upper limits of lipid accumulation—critical traits that conventional reviews have largely failed to address through systematic comparative analysis. This review adopts “eukaryotic-prokaryotic parallel comparison” as its overarching framework, tracing the full-spectrum technological chain from photobiological hydrogen production and anaerobic biomethanation to liquid biofuels (bioethanol/biobutanol, biodiesel, and sustainable aviation fuel). The analysis reveals the distinct industrialization scenarios to which the two phylogenetic groups are respectively suited across different energy pathways. On this basis, the review examines the differentiated enabling effects of CRISPR-Cas9 gene editing and AI-driven optimization on the two platforms, and evaluates the prospects and constraints of algal biorefineries from three dimensions: multi-product co-production biorefining, wastewater treatment-carbon capture synergy, and techno-economic analysis coupled with life cycle assessment (TEA-LCA). It concludes that harvesting/dewatering energy consumption and photobioreactor capital expenditure remain core bottlenecks to economic viability, while the introduction of carbon trading mechanisms (carbon price at US50–100 per ton CO₂) could fundamentally restructure the cost architecture of algal fuels. Future efforts should prioritize low-energy harvesting technologies, cross-domain enzyme system transplantation, component-based fractional valorization, and ecological risk assessment of transgenic algae, so as to accelerate the transition of algal energy systems from laboratory research to industrial deployment.