2-Thiobarbituric acid (TBA) is one of the most polymorphic rich molecular solids currently known. Here, five most common polymorphs of TBA (form Ⅰ, Ⅱ, Ⅲ, Ⅳ and hydrate) were investigated using terahertz time-domain spectroscopy (THz-TDS). All results of the terahertz spectra showed that there were significantly different absorption features for polymorphs, which indicated that THz-TDS technique could effectively identify different types of TBA polymorphs. Form Ⅳ belongs to the tautomeric polymorph, and the absorption features of form Ⅳ at 0.65(broad band), 1.02 and 1.41 THz are significantly different from physical mixture of form Ⅰ and form Ⅱ. Density functional theory (DFT) calculation was performed to optimize two possible molecular structures and simulate the corresponding vibrational spectra. The theoretical result showed that simulated structure (form a) had absorption peaks at 0.41/0.47, 0.89 and 1.35 THz, which agrees with the THz experimental spectrum. Therefore, it is confirmed that, in the TBA form Ⅳ, the first hydrogen bond is formed between thione S7 of isomer A and amide H23 of isomer B in the structure of form Ⅳ, while the second one is constituted by amide H13 of isomer A and thione S20 of isomer B. The characteristic absorption modes of form Ⅳare also assigned based on the simulation results of DFT calculation.