The invention discloses a hybridoma cell strain AB2 for secreting an anti-amikacin monoclonal antibody and an application thereof and belongs to the technical field of immunochemistry. The hybridoma cell strain AB2 of the anti-amikacin monoclonal antibody is preserved in CGMCC with the preservation number of CGMCC No.13086. The monoclonal antibody secreted by the cell strain is measured by means of an indirect competitive-ELISA method, and IC50 to amikacin is 0.65mu g/L. The recovery rate range of amikacin in milk is 73.55-84.61%, and the recovery rate range of amikacin in eggs is 73.70-105.75%. The hybridoma cell strain AB2 provides a raw material for immunodetection of residual amikacin in food, and has actual application value.