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Therapy dogs have been increasingly incorporated into a variety of medical treatment programs to improve patients’ treatment outcomes and wellbeing. However, research investigating the stress level of therapy dogs in this setting is limited. This is the first randomized–controlled and prospective study that investigated the wellbeing of therapy dogs in an inpatient stroke rehabilitation program. In this study, 14 therapy dog–handler pairs were embedded in an inpatient stroke rehabilitation program to provide animal-assisted treatment (AAT). These therapy dog–handler pairs actively participated in stroke rehabilitation by walking with the patient, playing fetch with the patient, and being petted/brushed by the patient, amongst various other AAT activities. To measure canine stress responses during the rehabilitation sessions, salivary cortisol and oxytocin concentrations, heart rate and heart rate variability, tympanic membrane temperature, and a behavioral evaluation were recorded before and after interactions with the patient. The results demonstrated that therapy dogs had significantly decreased heart rate and increased heart rate variability after the AAT session. Right tympanic temperature significantly increased after the session, but there was no significant difference in terms of salivary cortisol or oxytocin levels, nor in stress-related behavioral evaluations after the AAT session. Taken together, the results suggest that incorporating AAT into an inpatient stroke rehabilitation program did not induce stress in the therapy dogs, and that the therapy dogs may have been more relaxed after the session.