2,4-Dichlorophenoxyacetic acid (2,4-D) is a common plant growth regulator, which can remain in food and, with long-term consumption, threaten human health. Therefore, it is necessary to propose an effective detection method. Terahertz time-domain spectroscopy technique (THz-TDS) has good advantages in the quantitative and qualitative analysis of most biomolecules due to its rich fingerprint characteristics. In this paper, density functional theory (DFT) was applied to geometry optimization and frequency vibration calculation of 2,4-D, and THz-TDS was used to quantitatively detect 2,4-D in Zizania latifolia. The results showed that there were three characteristic absorption peaks of 2,4-D at 1.36, 1.60, and 2.38 THz, respectively, and the theoretical spectra were in good consistency with experimental spectra, with slight discrepancies. Additionally, the absorption peak at 1.36 THz had the best absorption characteristics and was chosen as the main peak for 2,4-D quantitative analysis. It was demonstrated that the limits of detection (LOD) of 2,4-D in Zizania latifolia were found to be as low as 5%, the absorbance intensity at 1.36 THz showed a good linear relationship (R2 = 0.9854) with 2,4-D concentration from 5% to 30%, and the recovery was 93.29%–98.75%. Overall, this work enriched the fingerprint database of pesticide molecules on the basis of terahertz spectroscopy and could provide a technical support for the detection of 2,4-D in food by terahertz spectroscopy.