It has been assumed that stroking relieves stress responses in dogs, and dogs with the activation of the left-brain hemisphere (right-pawed) may show better adaptation to stress conditions. The aim of the study was to determine whether the stroking stimulus induced changes in the level of selected neuroregulators in dogs’ blood and whether these changes depended on the sex and the predominance of the activity of one of the brain hemispheres. The study involved 40 dogs of various breeds and both sexes. The experimental animals were subjected to a behavioral tests (Kong test), and the levels of noradrenaline, serotonin, and cortisol were determined in their blood plasma. The results of the behavioral test revealed that most dogs exhibited increased activity of the left hemisphere. Furthermore, irrespective of the sex and paw preference, stroking the animal was found to alleviate the stress response, which was reflected in reduced cortisol levels and increased serotonin levels. It was found that the plasma noradrenaline, cortisol, and serotonin levels were lower in the female dogs than in the males. Additionally, the plasma noradrenaline and serotonin levels were higher in the right-pawed dogs than in the left-pawed dogs. The present results confirm the assumption that right-pawed dogs adapt to stressful conditions more readily.