As a new beer fermentation technology, high temperature and high gravity fermentation has brought many benefits to brewery industry, but there are also a series of problems such as the decrease of yeast flocculation ability at the end of fermentation and the high concentration of higher alcohols. To increase yeast flocculation ability and reduce the production of higher alcohols in high temperature and high gravity fermentation of beer, BAT2 was replaced by the FLO5 expression cassette to obtain the mutant strain S6-BF2. Real-time quantitative PCR showed that the relative transcriptional level of FLO5 in S6-BF2 improved 17.8 times compared with that in S6. The flocculation ability of mutant S6-BF2 heightened by 63% compared to that of the original strain S6, and the concentration of higher alcohols decreased from 175.58 mg/L to 159.58 mg/L in high temperature and high gravity fermentation of beer. Moreover, the activity of mitochondrial branched-chain amino acid transferase was repressed, resulting in the production of higher alcohols of 142.13 mg/L, reduced by 18.4% compared to that of the original strain S6, meanwhile, the flocculation ability of mutant S6-BF2B1 kept unchanged compared to the mutant S6-BF2. The determination result of flavor compounds showed that the higher alcohols/ester ratio in beer was reasonable. This research has suggested an effective strategy for enhancing yeast flocculation ability and decreasing production of higher alcohols in high-temperature and high-gravity brewing.