Dense dislocation entanglement and cellular structures impart unprecedented mechanical properties to materials manufactured using laser powder bed fusion (LPBF). However, the interaction between substructures, twins, and dislocations during deformation remains a topic of debate. In this study, the intrinsic strengthening mechanisms and deformation behavior of LPBF twinning-induced plasticity (TWIP) steels subjected to various heat treatment methods were thoroughly investegated. The results reveal that the high strength primarily arises from dislocation strengthening, accounting for over 50 % of the yield strength in LPBF TWIP steels. Additionally, annealing at 400 degrees C preserves yield strength through stabilized dislocation configurations, whereas annealing at 600 degrees C, 800 degrees C, and 1050 degrees C leads to a substantial attenuation in dislocation density, thereby reducing yield strength. The high density of dislocations, coupled with pre-existing stacking faults (SFs), results in significant lattice distortion. Consequently, dislocation slip is hindered, raising the twinning stress and reducing twin thickness during deformation. In contrast to the hardening effect attributed to secondary twin variants after annealing at 800 degrees C, the as-built materials demonstrate ongoing softening, resulting in localized plastic deformation that concentrates at grain and twin boundaries until failure occurs. Furthermore, localized dislocation cross-slip generates steps and forms Lomer-Cottrell (L-C) locks in the as-built materials. This study provides valuable insights into the underlying mechanisms influencing the strength and ductility of LPBF TWIP steels.