A reliable and sensitive competitive real-time immuno-PCR (RT-IPCR) assay for the determination of anthracene (AN) was developed. 9-Anthracenebutanoic acid, gamma-oxo-(ANA) was synthesized as the hapten of AN. Mixed anhydride reaction (MAR) was used to couple the ANA to ovalbumin (OVA) to form artificial coating antigen. Active ester method (AEM) was used to couple the ANA to bovine serum albumin (BSA) to form artificial immune antigen. Male New Zealand white rabbits were immunized with immune antigen to obtain polyclonal antibodies (pAbs), with which, a novel RT-IPCR assay for determination of AN was described. Under optimized assay conditions, AN can be determined in the concentration range from 1 fgmL(-1) to 100 pgmL(-1) with a detection limit of 0.5 fgmL(-1). The cross-reactivities of the anti-AN antibody to seven structurally related compounds were below 15%. Environmental water samples were successfully analyzed, showing a good accuracy and suitability to analyze AN in field samples. Recovery was between 93.3% and 120.0% and would be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions.