Dimethomorph (DMM) is a widely used high-efficiency fungicide which poses unpredictable threats to the ecological environment and public health. It is significant to establish a sensitive and robust analytical method for DMM detection. Here, we fabricated a catalytic hairpin assembly (CHA) - based immunofluorescence (IMF) biosensor by using single-strand DNA and DMM antibody co-modified gold nanoparticles (H₀-Ab-Au) as anti-interference probes and DMM antigen coated 96-well plate as the immune recognition element and CHA reaction vessel. Parameters relevant to AuNP probes preparation and CHA reaction environment were optimized. After optimization, the LOD of 0.002 ng/mL was calculated, with a linear correlation in inverse proportion to DMM concentration ranging from 0.01 ng/mL to 50 ng/mL. In addition, the developed biosensor was successfully applied to a variety of complex matrix samples, with satisfactory recoveries over a range of 86.74%–118.60%. Moreover, the detection results of IMF biosensor have a good correlation with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Therefore, our proposed IMF biosensor exhibits ultra-high sensitivity and excellent specificity, as well as great potential for application to other hazards.