Background and Aims: Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) with a globally increasing prevalence, partially driven by alterations in gut microbiota. Although biological therapy is the first-line treatment for CD, a significant proportion of patients experience a primary non-response or secondary loss of response over time. This study aimed to explore the differences in gut microbiota among CD patients with divergent long-term responses to biological therapy, focusing on a long disease course. Methods: Sixteen CD patients who applied the biological agents for a while were enrolled in this study and were followed for one year, during which fecal specimens were collected monthly. Metagenomic analysis was used to determine the microbiota profiles in fecal samples. The response to biological therapy was evaluated both endoscopically and clinically. Patients were categorized into three groups based on their response: R (long-term remission), mA (mild active), and R2A group (remission to active). The differences in the gut microbiota among the groups were analyzed. Results: Significant differences in fecal bacterial composition were observed between the groups. The R2A group exhibited a notable decline in gut microbial diversity compared to the other two groups (p < 0.05). Patients in the R group had higher abundances of Akkermansia muciniphila, Bifidobacterium adolescentis, and Megasphaera elsdenii. In contrast, Veillonella parvula, Veillonella atypica, and Klebsiella pneumoniae were higher in the R2A group. Conclusions: Gut microbial diversity and specific bacterial significantly differed among groups, reflecting distinct characteristics between responders and non-responders.