There is growing concern that antibiotic use in livestock production is contributing to contamination of soil and aquatic environments. Monensin, a polyether ionophore antibiotic, accounts for approximately 13% of total subtherapeutic livestock antibiotic usage in the USA and has been widely detected in aquatic environments. Due to insufficient ultraviolet absorbance, liquid chromatography analysis of monensin in environmental samples is limited to equipment with mass spectrometry (e.g., liquid chomatography-mass spectrometry [LC-MS]). However, LC-MS equipment is costly, and extensive sample preparation and clean-up is often required. Rapid, low-cost analytical techniques are needed to monitor for monensin residues in the environment. In this study, a commercially available enzyme-linked immunosorbent assay (ELISA) for detecting monensin in animal feed extracts was evaluated for determination of monensin in water, soil, and manure. The monensin ELISA was highly sensitive, with limits of detection and quantification at 1.5 and 3.0 pg L-1, respectively. Recovery of monensin in spiked water samples was approximately 100%. Cross-reactivity was not observed with similar polyether ionophores, tetracyclines, macrolides, or sulfonamides. Concentrations of monensin using ELISA were greater than concentrations measured with LC-MS. This is attributed to cross-reactivity of the monensin ELISA toward structurally similar monensin compounds, such as factors (slight naturally produced structural variants) and metabolites. Results from this study showed that ELISA can be a reliable, rapid, and low-cost alternative to LC-MS analysis of environmental samples.