The invention relates to a double-cross-linking chemical synthesis method of a citrinin-protein-coupled antigen and a preparation method of an anti-citrinin monoclonal antibody, belongs to the field of biotechnology. The synthesis method of the citrinin-protein-coupled antigen comprises the following steps that: the citrinin and a double-cross-linking agent (C2H3O)-(CH2)n-(C2H3O) react at first and is then coupled with a carrier, glycine is used for occlusion after reaction, and separation and purification are carried out finally, thereby obtaining the citrinin-carrier-coupled antigen. The preparation method of the anti-citrinin monoclonal antibody comprises the following steps: carrying out cell fusion and screening with the hybridoma technique to obtain a hybridoma cell line of the anti-citrinin monoclonal antibody with stable secretion, preparing ascitic fluid, and purifying, thereby obtaining the anti-citrinin monoclonal antibody. The invention has the advantages of mild preparation conditions, simple operation and controllable process. The obtained anti-citrinin monoclonal antibody has high specificity and high affinity, and can be further used for preparation of the enzyme linked immunosorbent assay kit or test paper card for citrinin detection.