Abstract: Objective To investigate the biological markers and their clinical significance in diagnosis and differential diagnosis of inflammatory bowel disease (IBD) in children. Methods The study had 22 cases of IBD including 6 cases of ulcerative colitis (UC) and 16 cases of Crohn’s Disease (CD). Twenty-four children without IBD were selected as controls. The serum perinuclear anti-neutrophil cytoplasmic antibody (pNACA) was measured by indirect immune fluorescence method. The serum anti-saccharomyces cerevisiae antibody (ASCA) IgG and IgA, anti-B mannose glycoside antibody (AMCA) IgG, anti-B glycoside sugar shell antibody (ACCA) IgA, Anti-bacterial flagellin antibody (Anti-cBir1) IgG, and the fecal calprotectin (FC) were determined by Enzyme linked immunosorbent assay (ELISA). Results The positive rate of serum pANCA was 100% in 6 cases of UC while it was negative in CD cases and control, and there was significant difference among three groups (P < 0.01). In CD cases, both positive rate of serum ACCA IgA and that of Anti-cBir1-IgG were 62.5% and the positive rate of ACCA IgA was 37.5%. Meanwhile, all of them were negative in UC cases and control. There were significant differences among three groups (P<0.01). The positive rate of FC was 100% in children with IBD. It was significantly higher than the positive rate in control group, 54.2% (P < 0.01). Conclusion The serum pANCA is a specific index for the diagnosis of UC. The serum ACCA IgA, AMCA IgG, ASCA IgG and IgA, and Anti-cBir1 IgG were specific to some extent in the diagnosis of CD. Increased FC can reflect the activity of IBD, but cannot be used for the differential diagnosis of IBD and non IBD.