The economical bio‐butanol‐based fermentation process is mainly limited by the high price of first‐generation biomass, which is an intensive cost for the pretreatment of second‐generation biomass. As third‐generation biomass, marine macroalgae could be potentially advantageous for conversion to clean and renewable bio‐butanol through acetone‐butanol‐ethanol (ABE) fermentation. In this study, butanol production from three macroalgae species (Gracilaria tenuistipitata, Ulva intestinalis, and Rhizoclonium sp.) by Clostridium beijerinckii ATCC 10132 was assessed comparatively. The enriched C beijerinckii ATCC 10132 inoculum produced a high butanol concentration of 14.07 g L−1 using 60 g L−1 of glucose. Among the three marine seaweed species, G. tenuistipitata exhibited the highest potential for butanol production (1.38 g L−1). Under the 16 conditions designed using the Taguchi method for low‐temperature hydrothermal pretreatment (HTP) of G. tenuistipitata, the maximum reducing sugar yield rate of 57.6% and ABE yield of 19.87% were achieved at a solid to liquid (S/L) ratio of 120, temperature of 110°C, and holding time of 10 min (Severity factor, R0 1.29). In addition, pretreated G. tenuistipitata could be converted to 3.1 g L−1 of butanol using low‐HTP at an S/L ratio of 50 g L−1, temperature of 80°C (R0 0.11), and holding time of 5 min.