This study aims to investigate the effect of fructooligosaccharide (FOS) (0.5, 1, 2, 3, and 4%) supplementation on the growth and survival of Bifidobacterium breve and Bifidobacterium longum in glucose, fructose, lactose, and sucrose (2, 3, and 4%) systems with 24-h growth and 10-day survival assays at 37 °C. FOS supplementation showed a higher growth-promoting effect on B. longum than B. breve in various sugar systems. The highest percentage of increase in growth index, 78.5%, was observed with 4% sucrose supplemented with 0.5% FOS in B. longum. In comparison, the highest percentage increase in growth index, 5.6 and 6.6%, was observed in the presence of 2% glucose and 4% lactose supplemented with 0.5% FOS in B. breve. In survival assay, FOS supplementation (0.5–4%) in a 2% lactose system showed the highest positive effect on the cell viability of B. longum on day-10. As for B. breve, FOS supplementation (1 and 2%) in the 2% sucrose system showed the highest positive effect on the cell viability, followed by FOS supplementation (0.5, 3, and 4%) in 2% sucrose and FOS supplementation (3 and 4%) in 2% lactose on day-10. This study demonstrated that the efficacy of FOS supplementation was depended on its concentration, sugar system and its concentration, and Bifidobacterium strain.