This study attempted to evaluate the potential for biofilm formation and the probiotic properties of lactic acid bacteria (LAB) isolated from the Carassius auratus intestine and to determine their antibacterial activity against crucial injurious bacteria of fish. The biological features test was carried out to obtain LAB with well-resistant pH, trypsin, bile and antibiotic challenge, and biofilm formation capacity. The most promising LAB isolates, y11 and y78 were identified as Lactobacillus johnsonii and Weissella confusa using 16S ribosomal RNA gene sequencing. We discovered that oral LAB additives improved immunity in the C. auratus by increasing immune-related expression of immunoglobulin M (IgM), superoxide dismutase (SOD), alkaline phosphatase (AKP), and lysozyme (LYS) in serum and interleukins (IL-10 and IL-1β), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in organs. After the introduction of Aeromonas veronii TH0426 for 15 days, the survival rate of C. auratus was determined as follows: the protection rate of group y11 was 45 %, and group y78 was 65 % when challenged with 109 CFU/mL density. This experiment successfully selected two LAB strains as the bioprotective agents and provided theoretical support for further LAB application in aquaculture. •First, two strains of probiotics were screened, which were resistant to acid and alkali, bile and trypsin and had no adverse effects on fish.•Second, two strains of probiotics can improve the immune level and enhance the immune function of crucian carp.•With TH0426 as the attack strain, the protection of the experimental group could reach about 50 %, while all the control group died.