Abstract: To increase the content of 4-vinylguaiacol (4-VG), a health-promoting flavor compound in Baijiu, microbial strains with high-yielding 4-VG were isolated and its production was optimized for application in Baijiu fermentation. In this study, high-yielding 4-VG strains were screened from Shennongjia sauce-flavor Daqu through primary and secondary screening, followed by identification. The fermentation conditions for 4-VG production by the strain were optimized using single-factor and response surface methodology (RSM) methods, and the strain tolerance was further investigated. Ultimately, the selected strain was used for fermentation in Baijiu. The results showed that the strain of JP-2 exhibiting both high endogenous 4-VG production and efficient precursor conversion was obtained by screening and was identified as Cronobacter maloniacus. The optimal fermentation conditions for high-yielding 4-VG of C. maloniacus JP-2 were sorghum juice-based medium, a sucrose concentration of 26.90 g/L, an initial pH of 5.0, and a fermentation time of 72 hours. Under the optimized conditions, the content of 4-VG reached 4342.92±401.62 μg/L, representing a 190.45% increase compared to the pre-optimization level. The tolerance test indicated that C. maloniacus JP-2 could survive at the condition of 6% alcohol, pH4.0, 45 ℃, and 250 g/L glucose, respectively. Compared to the control group, 4-VG contents in fermented grains at the end of fermentation and Baijiu made with biofortification with C. maloniacus JP-2 increased by 53.85% and 279.80% (P<0.05), respectively. Additionally, the JP-2 inoculation also increased esters contents and improved sensory qualities, particularly enhancing caramel and floral-fruity notes in Baijiu. This study would provide excellent strains and a data basis for improving the quality and health benefits of sauce-flavor Baijiu.