We demonstrated an efficient screening method for rabbit scFv antibodies using antigen-coupled multi-lamellar vesicles (Ag-MLVs) as solid supports. Model phages displaying mouse anti-human IgG scFv at a probability of 10-10% were successfully isolated by Ag-MLVs after 3 or less rounds of biopanning, whereas they could not be isolated using conventional antigen-coated immunotubes. This screening method was applied to isolate rabbit antigen-specific scFvs from 4 different phage libraries. Biopanning procedures employing Ag-MLVs yielded positive phages in the 3rd round or earlier, and specific antigen-binding of scFvs was observed after the 1st round in two biopanning selections. The dissociation rate constants (k) of isolated scFv clones tended to decrease with progressing biopanning rounds. The average dissociation constants (K) of the isolated scFvs ranged between 1.7 and 87 nM, whereas the lowest K of 12 pM was recorded for anti-CRP scFv. Comprehensive characterization of 355 different clones of the isolated rabbit scFvs presented a relatively low isoelectric point, and most of these were more thermo-stable than the conventional mouse scFvs, based on their instability and aliphatic indices. These results clearly indicate the advantages and potential of a combination of rabbit scFv-displaying phage library and biopanning using Ag-MLVs for antibody discovery. In addition, the results obtained in this study support the suitability of rabbit scFvs for several applications, including the development of diagnostic agents and affinity ligands for molecular diagnosis and bioseparation.