<p><i>Burkholderia gladioli</i> pv. <i>cocovenenans</i> (<i>B. gladioli C.</i>) poses a threat to consumer health, making strengthen supervision of <i>B. gladioli C.</i> essential. A dual-readout lateral flow biosensor was described based on the combined colorimetric and magnetic properties of polydopamine-coated Fe₃O₄ magnetic nanospheres (Fe₃O₄@PDA MNSs). The biosensor utilized a pair of monoclonal antibodies (mAb) as specific recognition module for point-of-care detection of <i>B. gladioli C</i>. The mAbs demonstrated a limit of detection (LOD) of 4.17 × 10⁴ CFU mL^− 1 for <i>B. gladioli C.</i> via sandwich ELISA. The developed Fe₃O₄@PDA-based lateral flow assay (LFA) utilized immunomagnetic separation and enrichment capabilities of Fe₃O₄@PDA-mAb probe for sample pretreatment. This assay facilitated both colorimetric and magnetic analysis modes, allowing for a visual LOD of 1.0 × 10⁴ CFU mL^− 1 and a quantitative LOD of 2.32 × 10³ CFU mL^− 1. Compared with the gold nanoparticles-based LFA, this method achieved an approximately ninefold increase in detection sensitivity. Moreover, the assay exhibited reliable performance with recoveries ranging from 81.0% to 103.5% in rice noodle and milk samples. The validation with a panel of 20 milk samples further confirmed its potential for application to complex matrix. These results indicated that the proposed Fe₃O₄@PDA-based LFA offered a practical and rapid solution for screening <i>B. gladioli C.</i> contaminants in food safety application.</p> Graphical abstract <p></p>