Probiotics have received increasing attention for their potential to promote health through improving intestinal barrier, enhancing human immunity, lowering cholesterol, regulating blood sugar, anti-cancer, anti-depression and so on. The prerequisite for probiotics to exert these functions is their ability to adhere to and colonize in the host’s intestinal tract. However, probiotics are susceptible to the extreme environment of the gastrointestinal tract (low pH in gastric juice and bile salts in intestinal fluids), as well as harsh processing and storage conditions, which may compromise their viability. At present, one of the effective methods to improve the survival ability of probiotics in the intestinal tract is the encapsulation of probiotics. Additionally, there are challenges related to inadequate detection methods for quantifying probiotics that colonize the intestinal tract. Therefore, this paper elaborates on the significance and challenges of probiotics intestinal colonization, the challenges they face, and methods to improve the probiotics viability during gastro intestinal transfer. The review also discusses detection methods for probiotics at different sites of intestinal tract. This review might show prospect for the in depth exploitation of probiotics used for improving intestinal health.