With the wide application as an alternative for perfluorooctane sulfonate (PFOS), perfluorobutane sulfonate (PFBS) has been frequently detected in the aquatic environment. However, the aquatic toxicity of PFBS is still poorly understood. The present work studied the aquatic toxicity of PFBS using freshwater algae Scenedesmus obliquus (S. obliquus) as indicator, and the toxicity of PFOS was also examined for comparison. The results showed that PFBS exhibited much lower toxicity to S. obliquus than PFOS. The EC₅₀ value was higher than 1800 mg L⁻¹ after 7 days of exposure to PFBS. By contrast, a much lower EC₅₀ value of 136.69 mg L⁻¹ was obtained for PFOS. Photosynthetic efficiency analyzed by chlorophyll fluorescence also verified that PFOS induced a higher toxic effect on the algae than PFBS. The malondialdehyde, catalase and superoxide dismutase results indicate that PFOS exposure led to the accumulation of ROS, which caused oxidative damage to the algae, thereby resulting in the inhibition in the growth and photosynthesis of the algae. Furthermore, transcriptome analysis indicates that the significant down-regulation of key genes related to photosynthesis induced by PFOS was the fundamental mechanism for the inhibition in photosynthetic efficiency and biomass growth of S. obliquus.