The use of probiotics has recently become a considerably promising research area. The most advanced fourth-generation probiotics involve beneficial bacteria enclosed in biofilms. However, differences in the effects of probiotics in biofilm and those in planktonic states are, as yet, unclear. In this study, it was ascertained that the biofilm mode of Lactobacillus paraplantarum L-ZS9 had a comparatively higher density and stronger resistance. Untargeted metabolomics analysis suggested a significant distinction between planktonic and biofilm cells, with amino acids and carbohydrate metabolism both more active in the biofilm mode. Furthermore, the in vivo experiment showed that the biofilm strain displayed better immunomodulation activity, which could increase the relative abundance of Lactobacillus in the intestinal microbiota of dogs. The relative abundance of intestinal microbiota participating in carbohydrate metabolism was higher in the biofilm probiotic-treated dogs. Correlation analysis between L-ZS9-producing metabolites, dog intestinal microbiome diversity and dog blood immune indexes (sIgA or IgG) revealed the interaction between these three components, which might explain the mechanisms by which biofilm L-ZS9 regulated the intestinal microbiome and immunity activity of the host, through the production of various metabolites. Findings of this study will, thus, enhance understanding of the beneficial effects of biofilm probiotics, as well as provide references for further investigation.