Little information is available regarding polyphenol variations in the food processing of edible and medicinal red seaweed, Betaphycus gelatinum. This study investigated the effects of Lactobacillus brevis fermentation on total polyphenol content (TPC), polyphenol profile, and antioxidant activity in Betaphycus gelatinum pretreated by ultrasound-assisted mild acid hydrolysis for the first time. During 60 h of fermentation, the viable colony number significantly increased, pH significantly decreased, and reducing sugar content significantly decreased initially, then significantly increased. Free TPC significantly increased to 865.42 ± 29.29 μg GAE/g DW (163.09% increase) with increasing antioxidant activity, while bound TPC significantly decreased to 1004.90 ± 87.32 μg GAE/g DW (27.69% decrease) with decreasing antioxidant activity. Furthermore, 27 polyphenol compounds were identified by ultra-high-performance liquid chromatography with Xevo triple quadrupole mass spectrometry. In total, 19 and 23 free polyphenols and 24 and 20 bound polyphenols were identified before and after fermentation, respectively. Before fermentation, bound trans-cinnamic acid (56.75%), bound rosmarinic acid (26.62%), and free trans-cinnamic acid (3.85%) were the main components. After fermentation, free rosmarinic acid (43.57%), bound trans-cinnamic acid (15.19%), bound rosmarinic acid (13.33%), and free trans-cinnamic acid (5.99%) were the main components. These results provide information for the food processing of Betaphycus gelatinum.