The effect of substrate bias on the degradation during applying a negative bias temperature (NBT) stress is studied in this paper. With a smaller gate voltage stress applied, the degradation of negative bias temperature instability (NBTI) is enhanced, and there comes forth an inflexion point. The degradation pace turns larger when the substrate bias is higher than the inflexion point. The substrate hot holes can be injected into oxide and generate additional oxide traps, inducing an inflexion phenomenon. When a constant substrate bias stress is applied, as the gate voltage stress increases, an inflexion comes into being also. The higher gate voltage causes the electrons to tunnel into the substrate from the poly, thereby generating the electron–hole pairs by impact ionization. The holes generated by impact ionization and the holes from the substrate all can be accelerated to high energies by the substrate bias. More additional oxide traps can be produced, and correspondingly, the degradation is strengthened by the substrate bias. The results of the alternate stress experiment show that the interface traps generated by the hot holes cannot be annealed, which is different from those generated by common holes.