Sargassum fusiforme polysaccharides (SFPs) have multiple activities. The fermentation of S. fusiforme by Lactobacillus can alter its polysaccharide properties and biological activities. In this study, three different Lactobacillus species (Lactobacillus plantarum (LP), Lactobacillus acidophilus (LA), and Lactobacillus rhamnosus (LR)) were selected to ferment S. fusiforme. The polysaccharides SFP (unfermented) and FSFP (fermented by LP, LA, or LR denoted as LP-SFP, LA-SFP, and LR-SFP, respectively) were extracted, and their physicochemical properties and biological activities were investigated. According to the results, fermentation caused significant changes in the physicochemical properties and biological activities of SFP. Specifically, FSFP showed a significant increase in uronic acid and fucose content and a significant decrease in molecular weight; LA-SFP and LR-SFP had stronger DPPH scavenging abilities; LR-SFP had the strongest inhibition of ROS production and cell mortality; LP-SFP and LR-SFP significantly increased SOD activity in zebrafish; LA-SFP had a significant effect on the proliferation of Lactobacillus plantarum; LP-SFP had a significant effect on the proliferation of Lactobacillus rhamnosus; and LA-SFP had a stronger food-excretion-promoting activity. In conclusion, the fermentation of Lactobacillus for the preparation of SFPs can change the physicochemical properties of polysaccharides and has broad potential for improving their biological activity.