ABSTRACT De-excitation gamma-ray lines, produced by nuclei colliding with protons, provide information about astrophysical environments where particles have kinetic energies of 10–100 MeV per nucleon. In general, such environments can be categorized into two types: the interaction between non-thermal MeV cosmic rays and ambient gas, and the other is thermal plasma with a temperature above a few MeV. In this paper, we focus on the latter type and investigate the production of de-excitation gamma-ray lines in very hot thermal plasma, especially the dependence of the line profile on the plasma temperature. We have calculated the line profile of prompt gamma rays from 12C and 16O and found that when nuclei have a higher temperature than protons, gamma-ray line profiles can have a complex shape unique to each nucleus species. This is caused by anisotropic gamma-ray emission in the nucleus rest frame. We propose that the spectroscopy of nuclear de-excitation gamma-ray lines may enable to probe energy distribution in very hot astrophysical plasmas. This diagnostics can be a new and powerful technique to investigate the physical state of a two-temperature accretion flows onto a black hole, especially the energy distributions of the protons and nuclei, which are difficult to access for any other diagnostics.