A rapid and quantitative technique is urgently needed in detecting toxicological and carcinogenic polycyclic aromatic hydrocarbons (PAHs) in the environment. Using a molecular beacon (MB), this study aimed at detecting the presence of naphthalene through an assay developed via a highly sensitive and robust, real-time fluorescent quantitative immuno-PCR (FQ-IPCR), which was then performed on serial dilutions of known naphthalene concentrations equivalent to 10-fold dilutions of 1-10(4) fg/mL. A correlation coefficient of 0.996 was identified, and a linear relationship between 1 fg/mL and 10 pg/mL, with y = 1.392x + 11.188, was obtained. A trace amount (1 fg/mL) of naphthalene congeners could be detected using this method. Five water samples were then used for validation, the results of which were further confirmed through a conventional enzyme-linked immuno sorbent assay (ELISA). Based on sensitivity and reproduction, the MB-based FQ-IPCR technique is a promising tool for monitoring environmental endocrine disruptors.