Avian influenza (AI) viruses pose a risk to the worldwide poultry industry, and the H9N2 AI virus has a profound immunosuppressive effect. The utilization of natural substances as immunostimulants is part of a global initiative to control AI viruses. Therefore, the goal of the present study was to examine if varying doses of the cyanobacterium Spirulina extract combined with the H9N2 vaccine would produce a greater immunological response. Thus, a total of 150 specific pathogen-free (SPF) chickens were allocated into six groups, 25 birds each, as follow: G1, G2, and G6 were supplemented with 200, 400, and 400 mg Spirulina extract/kg feed, respectively, whilst the feed in G3, G4, and G5 were not supplemented with Spirulina extract. At 21 days old, only the chickens in G1, G2, and G3 were vaccinated with the H9N2 AI vaccine. After four weeks post-vaccination, the chickens in G1, G2, G3, G4, and G6 were challenged with H9N2 AI Egyptian strain. The challenged virus was selected from a recent circulating Egyptian strain during 2022, and it was related to A/quail/Hong Kong/G1/97-like virus lineage and clustered with sub-lineage EGY-2. It had a high identity percentage of 92.6% with the A/chicken/Iran/av1221/1998 (Boehringer Ingelheim) vaccine. The results of reverse transcriptase real-time- polymerase chain reaction (rRT-PCR) revealed that no shedding of the virus was reported only in G1, G2, G3, and G5. However, chickens in Group 6 that were not vaccinated but were fed 400 mg of Spirulina extract/kg of feed only reduced but did not prevent virus shedding, as was the case in G1, and G2. The supplementation of Spirulina extract in low (200 mg/kg of feed) and high (400 mg/kg of feed) concentration with the birds vaccinated with H9N2 AI vaccine (G1 and G2) induced prominent immuno-stimulatory effect in a dose dependent manner where it strongly enhanced the phagocytic percent of broilers' peripheral blood monocytes, phagocytic index, and lysozyme at all days post vaccination (dpv) and days post challenge (dpc) compared to other groups with significant differences at 21st dpv, 28th dpv, 7th dpc,and 14th dpc, respectively. The supplementation with Spirulina extract in G1 and G2 induced the highest hemagglutination inhibition antibody titer in a dose-dependent manner at all time intervals. The antibody titer post-vaccination was significantly increased in G1 and G2 at 14th, and 21st dpv, respectively in comparison with G3. Furthermore, G1 and G2 showed higher significant antibody titers at 7th and 14th dpc, respectively compared to G3, G4 and G6. Furthermore, Spirulina extract (200 and 400 mg/kg feed) in G1 and G2 showed anti-inflammatory effect by downregulating nitric oxide levels at all times post-challenge with a significant difference at 3-7 days post-challenge compared to other groups, with improved histopathological alterations in the trachea, lung, kidney, spleen, and bursa of Fabricius. In conclusion, vaccination in conjunction with either dose of Spirulina extract (G1, and G2) prevents viral shedding, increases the immune response, and reduces inflammation and histopathological change caused by H9N2 AI infection in dose dependent manner. We recommend the use of 400 mg Spirulina extract/kg feed as a natural immunostimulant in conjunction with the H9N2 vaccine to achieve the highest possible level of protection against H9N2 AI infection.