As kanamycin (KAN) residue in animal products is harmful to consumers, a rapid and sensitive method for KAN detection needs to be established. KAN monoclonal antibody (KAN-mAb, 1D11) with the half maximal inhibitory concentration of 1.16 ng/mL was prepared in this study. A one-pot method was used to synthesize Fe3+-chelated polydopamine nanospheres (Fe@PDANs) with excellent characteristics of strong light absorption. The novel label of Fe@PDANs and KAN-mAb was used to develop a lateral flow immunoassay (LFIA) for the sensitive detection of KAN. The limit of detection of the Fe@PDANs-based LFIA (Fe@PDANs-LFIA) for KAN was 0.0191 ng/mL, which was 2.75 times lower than PDANs-based LFIA. Furthermore, the Fe@PDANs-LFIA was successfully applied to detect KAN in pork, milk, and honey samples, with recoveries ranging from 93.75% to 113.80% (coefficient of variation < 10%). Therefore, Fe@PDANs have potential for the detection of analytes in LFIA.