An enzyme‐linked immunosorbent assay (ELISA) for 2,4‐dichlorophenoxy acetic acid (2,4‐D) was developed using polyclonal antibodies raised in sheep, immunized with a derivative of 2,4‐D (2,4‐dichlorophenoxy acetyl‐2‐succinamyl hydrazine) conjugated to keyhole limpet haemocyanin. The mean percentage recovery of 2,4‐D from fortified water samples was 104% and ranged from 96 to 120% for fortified orange juice. The sensitivity of the assay without concentration of sample or sample clean‐up was 0.2 ng ml−1for water and 50 ng ml−1for orange juice. With dichloromethane extraction of 2,4‐D from orange juice, 1 ng ml−1could be detected. 2,4,5‐T was the only herbicide investigated which cross‐reacted significantly with the 2,4‐D antisera. The ELISA also detected 2,4,5‐T in water, being sensitive to 0–66 ng ml−1. The ELISA is suitable for direct screening of water and fruit juice samples for 2,4‐D contamination.