Aims To determine the relationship between adhesive ability of probiotics and acidic residues in human colonic mucin, we developed a new screening method using Biacore to evaluate adherence of bacteria before and after sialic acid or sulphate residues were blocked or removed from mucin. Methods and Results Ten strains of lactobacilli and three strains of bifidobacteria isolated from human faeces were evaluated for their adhesive properties to soluble human colonic mucin (sHCM) using the Biacore binding assay. Three strains (Lactobacillus strain ME-522, Lact.gasseri ME-527 and Bifidobacterium bifidum MCC1092) showing significant adherence were selected. Decreased binding activities were observed after removing sialic acid of sHCM using sialidase. However, after removing the sulphate residue using sulphatase, the adhesion of ME-527 decreased; whereas the remaining two strains had increased adhesion. The adhesion of three probiotics significantly decreased after the sulphate residue was blocked by elution with barium chloride. Conclusions A new evaluation method using the Biacore assay was developed to observe binding properties to the acidic residues of sHCM. Results indicated that there was a strong relationship between probiotic adhesion and acidic residues of sHCM. Significance and Impact of the Study This is the first report showing a screening method that quantitatively measures the binding between bacteria and acidic residues in sHCM using the Biacore binding assay; and provides a new method for the selection of probiotics in the future.