Abstract: Effective supplementation of probiotics can be beneficial to intestinal health, but in situ analysis of probiotics activity has rarely been reported. In this study, by coupling fluorescein 5-isothiocyanate (FITC) and 5(6)-carboxytetramethylrhodamine N-succinimidyl ester (5(6)-TAMRA-SE) with D-lysine, two fluorescent D-amino acids (FDAAs) probes were obtained:green probe (fluorescein-D-lysine, FDL) and red probe (TAMRA-D-lysine, TDL). Then, we tried to label the three kinds of probiotics, Lactobacillus acidophilus (LA), Lactobacillus casei (LC) and Veillonella atypica (VA) in vitro. FDAAs was applied to the labeling of intestinal flora in mice, and a method was established to investigate the oral survival rate of three commonly used probiotics. All animal experiments were approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine. The results show that the two synthetic FDAAs can be non-toxic and 100% for the in vitro labeling of the three probiotics. Known by FDAAs two-step labeling of oral probiotics, the high survival rate of LA was 92.30% ±1.67%. The survival rates of VA and LC are similar, 84.13% ±4.06% and 82.27% ±2.43%, respectively. This study can quickly compare the changes of colonization survival rate of different probioticsin vivo, provide theoretical support for the in situ colonization activity of probiotics in the intestine, and guide the rational drug use of clinical probiotics.