Surfactin is a promising microbial lipopeptide with wide applications in food, environmental, agricultural, and pharmaceutical fields. However, its high cost caused by low productivity largely limits the commercial application. In this study, genome shuffling was employed to improve surfactin production in Bacillus velezensis strain LM3403 via recursive protoplast fusion. RT-qPCR analysis was employed to evaluate the transcriptional variance of surfactin synthase genes and surfactin efflux gene to insight into the variance underlying the recombinant strain. After three rounds of genome shuffling, a high-yield and genetic stable recombinant F34 was obtained, exhibiting dramatic improvement in surfactin production (from 229.60 ± 7.10 mg/L to 908.15 ± 5.65 mg/L) with high proportion of long carbon chain homologues. Scale-up fermentation confirmed that F34 had good growth performance and reached the yield of 917.05 ± 10.25 mg/L in a 30 L fermenter, which was 3.99-fold that of the initial strain. RT-qPCR analysis showed that the transcriptional levels of surfactin synthase genes srfAA and sfp, and surfactin efflux gene swrC in F34 were 8.12-fold, 9.27-fold, and 8.45-fold higher than those of LM3403, respectively. The upregulation of genes were consistent with the high surfactin yield in F34, indicating the increased capability of surfactin biosynthesis and transmember efflux in F34. To our knowledge, this is the first attempt to employ genome shuffling to breeding a B. velezensis strain to improve surfactin yield. The research helps us to understand the mechanisms underlying surfactin overproduction and provide references for further rational strain improvement.