Ergosterol Peroxide is a steroid derivative with various biological properties such as anti-cancer, anti-inflammatory, etc. It also has certain antibacterial activity in marine ecosystems. Therefore, analyzing the structure of ergosterol peroxide is crucial for exploring its activity mechanism. As an important quantum chemical calculation method, density functional theory has been increasingly applied in predicting the structure, energy, frontier molecular orbitals, and organic structure spectroscopic analysis of molecules. In this work, the spatial structure of ergosterol peroxide molecular was constructed using GaussView 6.0 software. Based on the density functional theory DFT-B3LYP method, the initial structure of ergosterol peroxide was initially optimized using the 3-21G basis set in Gaussian 09W software. Based on the coarse optimized structure, the structure was further optimized using the 6-311++G (d, p) basis set to obtain the molecules most stable configuration, energy, and frontier orbital distribution. Then, based on optimizing the structure, the theoretical infrared (IR) and Raman spectra of ergosterol peroxide were calculated using the 6-311G basis set. The error frequency correction factor of the theoretical calculation results was selected as 0.961 3 for correction. The experimental IR and Raman spectra of ergosterol peroxide solid powder were measured using experimental methods. From theoretical calculations and experimental results, it can be seen that in the theoretical infrared spectrum, ergosterol peroxide moleculesmainly exhibit significant vibrations in the range of 3 700～2 800 and 1 500～600 cm-1. The former mainly exhibits stretching vibrations, and the latter contains multiple vibrations. The characteristic peak frequency wavenumber error of both theoretical and experimental infrared spectra isless than 30 wavenumbers, indicating that the theoretical calculation results are relatively reliable. The corresponding bands in the theoretical Raman spectrum from 2 966 to 2 879 cm-1 and the experimental spectrum from 2 978 to 2 856 cm-1 are assigned to C—H stretching vibration characteristic peaks. The peak positions in the theoretical Raman spectrum are slightly blue-shifted compared to the experimental spectrum, but overall, they agree well. This study analyzed the optimal structure, frontier molecular orbitals, and vibration spectra of ergosterol peroxide, providing a theoretical basis for vibration spectrum detection and structure identification. Fundamental structural and spectral data were provided to further explore the application of ergosterol peroxide in marine ecosystems and the pharmaceutical field.