Paraquat (PQ) poisoning is a serious threat to human health that leads to pulmonary toxicity, neurotoxicity, and inflammation. Protecting humans from PQ exposure requires the development of rapid analytical methods for on-site detection. Here, two monoclonal antibodies against PQ were generated and an immunochromatographic assay (ICA) was exploited to determine PQ concentrations in water samples. The results showed that the monoclonal antibody 1D6 exhibited higher affinity and sensitivity, with an affinity constant of 5.4 × 108 mol/L and a limit of detection as low as 0.02 ng/mL. Without sample pretreatment, the developed ICA method provided visible limits of detection ranging from 0.25 to 1 ng/mL, and cut-off limits ranging from 1 to 5 ng/mL, where average recoveries were between 83.15% ± 1.9% and 94.49% ± 2.45% with a coefficient of variation ranging from 1.40% to 7.37%. Importantly, these observations were consistent with liquid chromatography tandem mass spectrometry. These data and results suggested that the ICA method was a reliable, portable, and high-throughput method for determining PQ residues in water samples.