We hypothesized that lameness has an impact on milk flow traits. The aim of the current study was therefore to investigate the relation between lameness and milk flow traits in dairy cows. For this study 73 healthy and 55 cows with lameness were selected. Lameness was diagnosed by a local specialized veterinarian, according to the standard procedure. The blood samples were collected during clinical examination. The milking properties of cows were evaluated twice in a row—during evening and morning milking. The selected cows in the current lactation did not receive veterinary treatment, and correct hoof trimming was not performed at least four weeks before the experiment. The measurements were taken by two electronic mobile milk flow meters (Lactocorder®®, WMB AG, Balgache, Switzerland). Milk flow data were processed using LactoPro 5.2.0 software (Biomelktechnik Swiss). Cortisol concentration was measured with the automated analyzer TOSOH®® AIA-360 (South San Francisco, CA, USA). We found out that milk flow traits can act as biomarkers of lameness in dairy cows. We determined that the milk yield in the first minute of healthy dairy cows was 1.77 kg higher than that of lame cows. The electrical conductivity during the initial time of milking of healthy cows was 0.24 mS/cm lower than that of the lame group. The milking duration of LA cows was 1.07 min shorter and the time of incline in milk flow from 0.5 kg/min till the reach of the plateau phase was longer. The risk of lameness was most clearly indicated by an increase in blood cortisol concentration; if its blood level in cows exceeds 1 µg/dL, the risk of identifying lameness increases 4.9 times.