Experiments were conducted for the study of the effect of cysteine addition on the microstructure of nanocrystalline Ni films electrodeposited from a nickel sulfate-based bath. Furthermore, the thermal stability of the nanostructure of Ni layers processed with cysteine addition was also investigated. It was found that with increasing cysteine content in the bath, the grain size decreased, while the dislocation density and the twin fault probability increased. Simultaneously, the hardness increased due to cysteine addition through various effects. Saturation in the microstructure and hardness was achieved at cysteine contents of 0.3–0.4 g/L. Moreover, the texture changed from (220) to (200) with increasing the concentration of cysteine. The hardness of the Ni films processed with the addition of 0.4 g/L cysteine (∼6800 MPa) was higher than the values obtained for other additives in the literature (<6000 MPa). This hardness was further enhanced to ∼8400 MPa when the Ni film was heated up to 500 K. It was revealed that the hardness remained as high as 6000 MPa even after heating up to 750 K, while for other additives, the hardness decreased below 3000 MPa at the same temperature.