This study describes a sensitive, competitive indirect ELISA for the detection of the antimicrobial triclocarban (TCC). The haptens were synthesised by derivatising the para position of a Ph moiety of TCC. The rabbit antisera were screened and the combination of antiserum 1648 and a heterologous competitive hapten containing a piperidine was further characterised. The IC50 and detection range for TCC in buffer were 0.70 and 0.13-3.60 ng/mL, respectively. The assay was selective for TCC, providing only low cross-reactivity to TCC-related compounds and its major metabolites except for the closely related antimicrobial 3-trifluoromethyl-4,4′-dichlorocarbanilide. A liquid-liquid extraction for sample preparation of human body fluids resulted in an assay that measured low part per billion levels of TCC in small volumes of the samples. The limits of quantification of TCC were 5 ng/mL in blood/serum and 10 ng/mL in urine, respectively. TCC in human urine was largely the N- or N’-glucuronide. The authors concluded that the results showed that the TCC concentrations of biosolids measured by the ELISA were similar to those detected by LCMS/MS. This immunoassay can be used as a rapid, inexpensive, and convenient tool to aid researchers monitoring human/environmental exposure to TCC to better understand the health effects.