Since ulcerative colitis (UC) has become a global concern, Lactiplantibacillus is considered an effective, safe strategy for alleviating intestinal inflammation in UC patients. The most advanced fourth-generation probiotics involve beneficial bacteria enclosed in biofilms with multiple advantages. However, the difference between the effect of probiotic biofilm and planktonic cells on UC remains unclear. This study indicated that the biofilm cells of Lactiplantibacillus LR-1 were more successful in increasing the colon length, relieving intestinal inflammation, and repairing colon damage, regulating the host immunity than the planktonic cells. Furthermore, the LR-1 biofilm cells helped prevent a decline in the Eubacterium hallii and Salinimicrobium levels and increased Kocuria and Candidatus Bacilloplasma abundance. Untargeted metabolomics analysis results suggested that the LR-1 biofilm was more successful in promoting the intestinal metabolism recovery of the UC mice than the planktonic cells. Finally, the phenotype-microbiota-metabolism network was conducted to reveal the relationship between these factors.