A new enzyme-linked immunosorbent assay (ELISA) for atrazine was developed based on covalent bonding of the small molecule hapten, 2-mercaptopropionic acid-4-ethylamino-6-isopropylamino-1,3,5-triazine (MPA–atrazine), to urea-glutaraldehyde (UGA)-treated microtiter plates. In this assay, the microtiter plate surface was treated with the UGA network to both introduce amino groups, which were used to cross-link with the hapten carboxylate groups, and efficiently prevent non-specific adsorption of antibodies, which successfully eliminated the time-consuming routine blocking step. Compared with HNO3-H2SO4-APTES-hapten coated ELISA (modified with a HNO3-H2SO4-APTES mixture and covalent-linked hapten) and conventional ELISA (coated with hapten–carrier protein conjugates), the novel ELISA format increased the sensitivity by approximately 3.5-fold and 7.5-fold, respectively, and saved 2.5 h and 34 h of coating hapten time, respectively. The method's 50% inhibition concentration for atrazine was 5.54 ng mL− 1, and the limit of detection was 0.16 ng mL− 1 after optimization of reaction conditions. Furthermore, the ELISA was adapted for analysis of atrazine in corn, rice, and water samples, demonstrating recoveries of 90%–108%. Thus, the assay provides a convenient alternative to conventional, laborious immunoassays for routine supervision of residue detection in food and the environment.