Cortisol, an essential glucocorticoid hormone, is crucial in regulating the stress response and maintaining physiological and behavioral homeostasis in mammals, including dogs. This review explores cortisol’s physiological and behavioral role in canines, focusing on its effects on stress, immune function, and metabolism. Various methods of measuring cortisol levels in dogs, invasive (blood, saliva, urine) and non-invasive (hair, fecal assays), are discussed regarding their accuracy and practical applications. The review also highlights the influence of different environmental factors, such as shelter conditions, human interaction, and music, on cortisol levels in dogs. Furthermore, the clinical and behavioral implications of abnormal cortisol levels are examined, with particular attention to conditions like Cushing’s disease and stress-related behavioral issues. The findings emphasize the importance of cortisol monitoring in veterinary practice and animal welfare, proposing future research directions to improve canine health and stress management.