A real-time fluorescent quantitative immuno-polymerase chain reaction (RT-IPCR) assay was developed for the detection of non-dioxin-like polychlorinated biphenyl (PCB) congener in soil samples. Based on the construction of 3,4-dichlorinated biphenyl (IUPAC PCB12) hapten and its immunogen, the specific polyclonal antibodies (pAbs) to PCB12 was obtained and used to develop a direct competitive RT-IPCR assay. Using the optimized assay, a standard curve for PCB12 was prepared. The linear range for the determination of PCB12 was from 10.0 to 1.0 x 106 fg/mL with a correlation coefficient of 0.98 and a detection limit of 1.53 fg/mL. The RT-IPCR assays were tested for their cross-reactivity profiles using four selected congeners and four Aroclor products. The results for the soil samples correlated with the concentrations of PCBs obtained by gas chromatography/mass spectrometry. This highly specific, sensitive, and robust assay can be applied to on-site tests of PCBs and serve as a model for other pollutant immunoassays.