Nowadays, tendency to improve human nutrition and consume new healthful foods such as low-calorie and functional ones has been increased. In this study, effects of ratios of sucrose/ d -tagatose (100:0, 0:100, or 50:50) as well as type of commercial probiotic strains ( Lactobacillus acidophilus LAFTI L10, Lactobacillus casei LAFTI L26, Lactobacillus rhamnosus HN001, and Bifidobacterium animalis subsp. lactis LAFTI B94) on biochemical and microbiological characteristics, percent of residual sugar, color, and sensory attributes of synbiotic chocolate milk were investigated during 21 days of refrigerated storage (5 °C). The treatments inoculated with L. acidophilus , L. casei , L. rhamnosus , and B. lactis showed significantly higher biochemical and color changes compared to non-probiotic ones. The greatest viability at the end of storage was related to the treatment of d -tagatose with L. rhamnosus (T-R) as well as d -tagatose with L. casei (T-C). Although L. acidophilus , L. casei , and L. rhamnosus mostly tended to ferment d -tagatose, B. lactis did not substantially consume the mentioned sugar. In general, the treatments T-R, ST-R (sucrose and d -tagatose with L. rhamnosus ), T-B ( d -tagatose with B. lactis ), and ST-B (sucrose and d -tagatose with B. lactis ) were realized as the best ones in terms of probiotic viability, functional property of d -tagatose, and sensory attributes. In conclusion, d -tagatose could be successfully used as a natural sugar substitute with functional properties for probiotic chocolate milks enhancing their health benefits, but the proper selection of ratio of sucrose/ d -tagatose and type of probiotic strain is recommended.