The ban on antibiotics as feed additives requires modern intensive farming with more comprehensive diseases control approaches. Currently, synbiotics serve as promising alternatives to enhance growth performance and improve health in the poultry industry. In this research, we investigated beneficial effects of Lactobacillus reuteri (LR) with its combination of gluco-oligosaccharides (GlcOS) supplementation on growth performance and intestinal health of broilers. A total of 900 1-day-old male Lingnan yellow-feather broilers were randomly allocated into the control group (CON group, and two experimental groups feeding basal diet supplementing LR (LR group) and its combination with GlcOS (RG group), respectively. The findings indicated beneficial effects of growth performance in experimental groups (LR and RG groups), as evidenced by decreasing the feed-to-gain ratio (F/G) in both experimental groups (P < 0.05) and increasing the average daily gain (ADG) in the RG group (P < 0.05). Simultaneously, both experimental groups increased the villus height/crypt depth ratio (VH:CD) (P < 0.001). Furthermore, the RG group showed increased activity of digestive enzymes (P < 0.05) and upregulated mRNA expression of tight junction protein and transportation protein (P < 0.05), while decreased the serum levels of d-lactic acid and diamine oxidase (P < 0.05), suggesting the improvement of the nutrient digestion and absorption, as well as the mucosal barrier integrity. Moreover, increased abundance of beneficial bacteria, including Bacteroides, Muribaculaceae and Prevotellaceae_UCG-001 (P < 0.05), leading to a finely altered gut microbiome and metabolome. Collectively, the findings of this research revealed that dietary supplemented LR and its combination with GlcOS could enhance the intestinal morphology, digestion, absorption and barrier function, and improve the cecal microbiota structure and metabolic function finally achieving the effect of improving growth performance of broilers. Overall, the effect of the combination of LR and GlcOS was synergistic, providing a future alternative to antibiotics as growth promoter.