The banning of antibiotics in feed has been a global trend. Fermented soybean meal (FSM) is expected to be a suitable substitute for antibiotics because of its benefits to improve intestinal health levels and immune performance. This study aimed to investigate the effects of FSM supplementation on the growth performance, intestinal health, and immune performance of laying hens during the brooding period. Four hundred eighty healthy 1-day-old laying hens were divided randomly into 4 groups (6 replicates/group, 20 hens/replicate) and were fed with different experimental diets from the day 1 (D1) to D56: (1) Control diet (CON); (2) Chlortetracycline (CTC; A diet supplemented with 0.5g chlortetracycline per 1kg of feed); (3) 4% fermented soybean meal (4% FSM; A diet containing 4% fermented soybean meal); (4) 8% fermented soybean meal (8% FSM; A diet containing 8% fermented soybean mea). Feed consumption was determined and average daily gain (ADG), average daily feed intake (ADFI), and FCR were calculated. After the trial ended, we collected the hens’ samples of serum, jejunal tissue, and immune organ, which were detected and analyzed. Simultaneously, immunofluorescence and Western blotting were used to detect the critical protein expression of Wnt/β-catenin signaling and evaluate intestinal development. The results showed that the ADFI and ADG of hens were significantly enhanced by adding 4% or 8% FSM, and the same effect as the CTC group compared with the CON group (P < 0.05). Meanwhile, the group of 4% FSM or 8% FSM improved the integrity of jejunal structure and barrier, reflected in increased villus height, enhanced protein expression of absorptive cell (Villin), tight junction proteins (Occludin and Claudin-1), proliferative cell marker, progenitor cell marker (SOX9), and stem cell marker (Lgr5), which has the same effect with the CTC group. Furthermore, CTC or 4% FSM treatment group resulted in a remarkable increase in Wnt/β-catenin signaling proteins (including β-catenin, TCF4, c-Myc, and Cyclin-D1) compared with the CON group (P < 0.05). Moreover, the results of immune organs showed that compared with the CON group, 4% FSM and 8% FSM could significantly improve the spleen index and thymus index of laying hens during the brooding period (P < 0.05). In addition, the IgA, IgM, and IgG levels in the serum of 4% FSM or 8% FSM groups were significantly increased than in the CON group (P < 0.05).