Knee joint rotation center displacement can be estimated in vivo through the analysis of helical axis (HAs) dispersion. HAs can be analyzed during walking, providing information on joint stability. The study aim was to describe knee HAs dispersion during walking in dominant and non-dominant legs of young and elderly healthy subjects. Twenty young (YG: age 23.3 ± 2.4 years) and twenty elderly (EG: age 69.3 ± 4.6 years) healthy subjects were asked to walk on a treadmill at a self-selected speed with reflective markers placed bilaterally on thighs and shanks to detect HAs dispersion and knee kinematics with an optoelectronic system. HAs dispersion was described during the following four phases of gait cycle: (1) flexion from 95% of the previous gait cycle to 10% of the subsequent gait cycle, (2) extension from 10% to 40%, (3) flexion from 40% to 75% and (4) extension from 75% to 95% of the gait cycle. Mean Distance (MD) and Mean Angle (MA) were used as HAs dispersion indexes during each gait phase. Participants showed greater MD and MA in sagittal and frontal planes during the first and second phases. EG revealed higher MD (p = 0.001) and MA (p < 0.001) during the first phase and higher MA (p = 0.001) during the fourth phase in both dominant and non-dominant legs on the sagittal plane. HAs dispersion could be related to the amount of forces acting on knee (first two phases) and knee degeneration (elderly). These results may be used as reference data in further studies on HAs dispersion in presence of knee pathologies or after knee surgery or rehabilitation.