Organophosphate pesticides (OPs) are often used as insecticides and acaricides in agriculture, thus improving yields. OP residues may pose a serious threat, duetoinhibitionof the enzymeacetylcholinesterase(AChE). Therefore, a competitive bio-barcode immunoassay was designed for simultaneous quantification of organophosphate pesticide residues using AuNP signal amplification technology and Au@Pt catalysis. The AuNP probes were labelled with antibodies and corresponding bio-barcodes (ssDNAs), MNP probes coated with ovalbumin pesticide haptens and Au@Pt probes functionalized with the complementary ssDNAs were then prepared. Subsequently, pesticides competed with MNP probes to bind the AuNP probes. The recoveries of the developed assay were ranged from 71.26 to 117.47% with RSDs from 2.52 to 14.52%. The LODs were 9.88, 3.91, and 1.47 ng·kg, for parathion, triazophos, and chlorpyrifos, respectively. The assay was closely correlated with the data obtained from LC-MS/MS. Therefore, the developed method has the potential to be used as an alternative approach for detection of multiple pesticides.