Atopic dermatitis (AD) is a common skin disease in animals and several therapeutic trials with various drugs have been conducted for more effective management of AD. However, these trials have not been able to properly address all the aspects of AD management because of the lack of good efficacy or due to significant side effects of the drugs being tested. Synbio-glucan functional spray is a functional skin spray using Synbio-glucan composed of β-glucan and probiotics. We designed a functional spray composed of Synbio-glucan (patent application number:10-1805863), distilled water, glycerin, solubilizer, and 40% alcohol. We tested the efficacy and safety of the functional spray on six dogs with AD. The trial was conducted with the consent of the caregivers. The spray was applied to the skin lesions, including the trunk, axillae, inguinal region, or periocular areas, thrice a day for 30 days. To evaluate the efficacy of this functional spray, we assessed the pruritus visual analog scale (PVAS) and the canine atopic dermatitis extent and severity index (CADESI)-4. At the end of one month, the results clinical scores after functional spray treatment showed a significant decrease in the PVAS (p = 0.03) and CADESI-4 (p = 0.03) in all the subject dogs with AD. This study thus confirmed that the Synbio-glucan functional spray is efficacious and safe for the treatment of AD in dogs.