BackgroundAlginate oligosaccharide (AOS) holds great potential as a novel feed supplement in farm animals. However, the effects of AOS on chicken health and the underlying mechanisms are not fully understood. This study aimed to optimize the enzymatic preparation of AOS by using bacterial alginate lyases expressed in yeast, investigate the effects of the prepared AOS on the growth performance and gut health of broiler chickens, and reveal the underlying mechanisms.ResultsFive alginate lyases from bacteria were cloned intoPichia pastorisGS115 and the alginate lyase PDE9 was expressed at relatively high yield, activity and stability inP. pastoris. Animal trials were carried out using 320 1-day-old male Arbor Acres broilers (four groups; 8 replicates/group × 10 chicks/replicate) receiving either a basal diet or the same diet supplemented with 100, 200 and 400 mg/kg PDE9-prepared AOS for 42 d. The results showed that dietary supplementation of 200 mg/kg AOS displayed the highest activity in promoting the birds’ ADG and ADFI (P< 0.05). AOS ameliorated the intestinal morphology, absorption function and barrier function, as indicated by the enhanced (P< 0.05) intestinal villus height, maltase activity, and the expression ofPEPT,SGLT1,ZNT1, and occludin. AOS also increased serum insulin-like growth factor-1, ghrelin (P< 0.05), and growth hormone (P< 0.1). Moreover, the concentrations of acetate, isobutyrate, isovalerate, valerate, and total SCFAs in cecum of birds fed AOS were significantly higher than the control birds (P< 0.05). Metagenomic analysis indicated that AOS modulated the chicken gut microbiota structure, function, and microbial interactions and promoted the growth of SCFAs-producing bacteria, for example,Doreasp. 002160985; SCFAs, especially acetate, were found positively correlated with the chicken growth performance and growth-related hormone signals (P< 0.05). We further verified that AOS can be utilized byDoreasp. to grow and to produce acetate in vitro.ConclusionsWe demonstrated that the enzymatically produced AOS effectively promoted broiler chicken growth performance by modulating the chicken gut microbiota structure and function. For the first time, we established the connections among AOS, chicken gut microbiota/SCFAs, growth hormone signals and chicken growth performance.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s40104-023-00887-4.