Using density functional theory (DFT) with numerical atomic functions including dispersion corrections, we establish a series of energy changes and Gibbs free energies of in situ and ex situ substitution by ammonia on Ni-Mo-S nanocluster, which includes Ni-Mo-Edge, Ni-S-Edge and Corner-Site. The results show that the in situ substitution on all active sites on Ni-Mo-S is hard to happen. The -SH group and hydrogen atom on coordinately unsaturated sites (CUS) of Ni-S-Edge can form H2S under a moderate condition, whereas the sulfur atoms on Ni-Mo-Edge are rather difficult to form H2S. The ex situ substitution of sulfur atoms on both Ni-S-Edge and Ni-Mo-Edge is energetically favored; however, the substitution may only occur on Ni-S-Edge under low partial pressure of ammonia based on the steady state of Ni-Mo-S active phase. The orbital properties show the eigenvalue has increased after substitution, which indicates the decreasing of binding electron capacity. Furthermore, the HDS performance would drop remarkably attributed to steric hindrance and lacking of activated hydrogen after substitution.