The adverse health effects of both active and passive tobacco smoke have been well-known in humans for a long time. It is presumable that even pets, which intimately share the owner’s lifestyle, may be exposed to the same risks. This study aimed to detect and quantify cotinine (a metabolite of nicotine) in the serum and hair of dogs using a specific commercial ELISA immunoassay kit. A total of 32 dogs, 16 exposed and 16 unexposed to the owner’s smoke, were enrolled. The cotinine concentration was higher in the exposed than the unexposed group in both matrices (p < 0.001), with greater values in serum than in hair (p < 0.001). Exposed bitches had higher hair cotinine than male dogs (p < 0.001). Conversely, serum and fur cotinine concentrations were lower in female than male dogs of the unexposed group (p < 0.01). The exposure intensity, age, and weight of the dogs did not affect cotinine concentrations. A cut-off value of 2.78 ng/mL and 1.13 ng/mL cotinine concentration in serum and fur, respectively, was estimated to distinguish between the exposed and unexposed dogs. Cotinine was confirmed as a valuable marker of passive smoking also in dogs. Although owners do not perceive secondhand smoke as a risk for their dogs, greater awareness should be advisable, especially in pregnant animals.