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Two-step indirect competitive enzyme immunoassay of DDT has been developed. The limit of detection and limit of quantification were 0.3 nmol·l−1 and 4.2 nmol·l−1, respectively. To shorten the analysis time, the one-step enzyme immunoassay has been developed by replacing anti-DDT antibody with the complex anti-DDT antibody-HRP. The anti-DDT antibody-HRP conjugate was prepared by periodate method. A concentration of the preparation was 1.9 mg·ml−1 and molar ratio Px/IgG was 1.87. The detection limit of the one-step ELISA reached 0.3 nmol·l−1, but the sensitivity of assay was very poor. The two-step enzyme immunoassay of DDT has been adapted for the chemiluminescent detection of the complex antigen-antibody. The detectability of chemiluminescent ELISA was comparable with that of chromogenic ELISA. A limited number of the artificially contamined samples have been tested. All samples were prepared by solid-phase extraction by using the commercial Agilent Zorbax SPE C18 columns. A background was observed in most tested samples. The recoveries obtained from the analysis of DDT spikes reached values between 89.0–161.0%, and 81.5–111.6% at standard addition 50 μg·kg−1, and 200 μg·kg−1, respectively. The overestimated amounts of DDT were determined in spinach and nectarines at standard addition 50 μg·kg−1.