Benzo[a]pyrene (BaP), a kind of polycyclic aromatic hydrocarbon, has great potential hazards to the ecosystem and human health. In this study, based on the preparation of BaP immunogen and coating antigen, a specific polyclonal antibody targeting BaP was generated, and a highly sensitive real-time immuno-PCR assay using the biotin-streptavidin system (BA-IPCR) was developed for detecting BaP in vegetables and barbecue food. Several detection conditions were optimized, such as blocking reagent, the concentration of streptavidin, and the concentration of biotinylated DNA (bio-DNA). Under the optimal conditions, a calibration curve was constructed in the concentration range of 5 pg L−1 to 10 ng L−1. The limit of detection was 2.85 pg L−1. Recovery of the spiked samples ranged from 92.1 to 109.6%. This immunoassay showed high specificity, with low cross-reactivity values with BaP analogues. The consistency between the results obtained from BA-IPCR and gas chromatography and mass spectrometer (GC-MS) was 98.96%. This proposed method is reliable and has great potential to detect trace BaP in environmental and food samples.