Qualitative and localized detection of oral probiotics in the host has great significance. In this study， a horizontal specific fluorescence in situ hybridization probe was designed to study the colonization of Lactobacillus rhamnosus in the host intestinal tract. Based on the genome sequences of L.rhamnosus Probio-M9 and other related species， the L. rhamnosus species-level specific probe （Probe-16S） and the L.rhamnosus Probio-M9 strain-level specific probe （Probe-SP） were designed. After these specific probes verified using 15 strains of Lactobacillus rhamnosus and 15 other probiotics of the genus， the L.rhamnosus Probio-M9 strain-level specific probe was examined its applicability both in vitro and in vivo by fluorescence in situ hybridization （FISH）. Finally， combining a fluorescent D-amino acid （FDAA）-based metabolic probe， the living cells of L.rhamnosus Probio-M9 were detected in the intestinal tract of rats. The strain-level FISH probe we designed could specifically identify the L.rhamnosus Probio-M9 from the gut microbiota. This method could combine with the FDAA probes to provide a reliable technic for further exploring the translocation of this probiotic strain in the host.