[Objective] To obtain anti-Listeria monocytogenes specific single-domain heavy chain antibody by subtractive panning, and to analyse the enrichment rules of specific phage clones in biopanning against composition diversity and complex antigens. [Methods] The whole cell of heat inactivated Listeria monocytogenes was used as antigen to screen specific single-domain heavy chain antibodies from an alpaca non-immune phage displayed library by solid panning technique. After 4 rounds of conventional panning and 1 round of subtractive panning, a total number of 384 phage clones randomly picked from each round were characterized by Phage-ELISA, the positive clones were sequenced and the binding specificity was analyzed by Phage-ELISA. [Results] Two anti-Listeria monocytogenes specific single-domain heavy chain antibodies were obtained. [Conclusion] Whole cell-based panning strategy was feasible for isolation anti-Listeria monocytogenes specific phage display antibodies under the optimized conditions in this study. Subtractive panning was efficient and necessary to eliminate non-specific clones.