Abstract: In order to improve the yield of γ-aminobutyric acid (GABA), strain CLYB1 was bred with ultraviolet mutagenesis and genome shuffling to produce high yield of GABA. After breeding, the hemolysis experiments and antibiotic sensitivity test were carried out on the recombinant strain. The strain CLYB1 was Bacillus velezensis, and the yield of GABA was 3.95 g/L. The GABA yield of CLYB1-Y breeding by ultraviolet mutagenesis from CLYB1 was 10.26 g/L, which was 160% higher than that of CLYB1. The GABA yield of CLYB1-YC breeding by genome shuffling from CLYB1-Y was 20.19 g/L, which was 411% higher than that of CLYB1. The hemolysis experiments and antibiotic sensitivity tests showed that CLYB1-YC had no hemolytic activity and was sensitive to 10 common antibiotics (penicillin, ampicillin, ceftriaxone, gentamycin, tetracycline, erythromycin, ciprofloxacin, lincomycin, chloramphenicol and puromyn). The Bacillus velezensis CLYB1-YC breeding by genome shuffling producing high γ-aminobutyric acid has a good application prospect.