Hemp seeds are the important resource of high-quality vegetable oil, while the numerous high-quality protein in the hemp seed meal has not been fully exploited. Hemp protein isolates (HPI) are the resource of the high-quality protein, containing 20 kinds of amino acids with a relative equilibrium of the content of 8 essential amino acid and a high content of Agr. However,the application of HPI in food processing was severely hindered by the poor protein solubility. At present, there was a few pieces of literature on the conformation of HPI, and the effect of pH change on its conformation during processing has not been reported. In this study, the conformation of hemp protein isolate (HPI) was characterized by Circular dichroism (CD) spectrum, Fourier transforms infrared (FTIR) spectroscopy, Fluorescence spectrum and Ultraviolet (UV) spectrum, which compared with soybean protein isolate (SPI). The effects of different pH conditions on the conformation of HPI were investigated. Conformation results showed that comparing with SPI, there was more α-helix content in HPI, determined by CD and FTIR, which was 39.1% and 33.1%, respectively, while SPI contained β-sheet. The exposure of aromatic amino acid residues from HPI was lower than SPI, revealed by intrinsic fluorescence and UV spectroscopy. Moreover, the content of disulfide bond of HPI (10.06 mmol·g^-1) was more than SPI, as was as higher surface hydrophobicity (374), but the value of free sulfydryl/total free sulfydryl of HPI was significantly lower than that of SPI. These results suggested that the tertiary structure of HPI was more compact than SPI, leading to a smaller particle size of nature HPI. The structure characteristics of HPI were changed in strong acid and strong alkaline conditions, which induced the structure of HPI to unfold and increased the size. As a result, the distance between aromatic amino acid residues was increased, and the energy transfer efficiency of Phe to Tyr and Trp was decreased. Finally, the fluorescence intensity of Try residues was decreased, and the fluorescence of Phe and Tyr residues was partly showed. Comparatively speaking, under acidic conditions, the conformation stability of HPI was relatively poor, and dissociation-association reactions were prone to the protein subunits. However, the conformation of HPI was relatively stable and with more flexibility. Therefore, with the help of fluorescence spectrum and UV second derivative spectrum, changes in the microenvironment of amino acid residues of HPI could be predicted, so as to understand changes in the three-dimensional structure of protein molecules. In conclusion, further understanding of HPI conformation and clarifying the influence of pH changes on HPI conformation are conducive to providing a theoretical reference for specific modification of HPI to prepare high-quality HPI and active physiological polypeptides.