The abusive use of 2,4-dichlorophenoxyacetic acid (2,4-D) was very common in the commercial production of bean sprouts. Herein, a simple and highly sensitive nano-SiO2-based ELISA method for residual 2,4-D detection was developed. The test depends on the direct competitive combination to anti-2,4-D antibody labeled by horseradish peroxidase (HRP) between nano-SiO2 immobilized by ovalbumin-2,4-D (SiO2-OVA-2,4-D) and free 2,4-D in sample. The linear range of this method was from 1 to 350 ng/mL (r = 0.997), and the limit of detection (LOD) was 0.079 ng/mL. The coefficient variations of intra-day and inter-day were all lower than 10% at three concentrations (20, 150, and 250 ng/mL). The mean recovery was 93.2%, and the specificity was satisfactory. The results indicated that the method could be useful for fast determination of residual 2,4-D in bean sprout samples using classic TMP/HRP system and a conventional UV spectrophotometer.