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<b>Background</b>: Vaccine Effectiveness (VE) provides an important indicator of vaccine performance under real-world conditions. However, evidence regarding influenza VE in Wuhan remains limited. This study applied a test-negative case-control design to estimate the effectiveness of influenza vaccination during the 2024-2025 influenza season in Wuhan. <b>Methods</b>: A test-negative case-control design was conducted among patients presenting with influenza-like illness (ILI) at outpatient and emergency departments of 41 healthcare institutions in Wuhan. All participants underwent influenza virus real-time reverse transcription polymerase chain reaction (RT-PCR) testing and were categorized as cases (RT-PCR positive) or controls (RT-PCR negative) based on laboratory results. <b>Results</b>: The analysis included 23,302 RT-PCR-confirmed influenza cases and 99,424 test-negative controls. The overall adjusted VE was 35% (95% CI: 30-40%). VE differed across age group, with higher estimates observed among adults aged 19-59 years (63%; 95% CI: 50-73%) and 60-69 years (60.7%; 95% CI: 46-72%), whereas lower effectiveness was observed among children aged 0.5-5 years (25%; 95% CI: 17-33%) and 6-18 years (25%; 95% CI: 14-36%). Similar protection was observed among individuals vaccinated in both the 2023-2024 and 2024-2025 seasons (42%; 95% CI: 28-54%) and those vaccinated in the 2024-2025 season only (40%; 95% CI: 36-45%), whereas VE was lower among individuals vaccinated only during the previous season (20%; 95% CI: 14-26%). VE also varied by vaccination timing, with the highest effectiveness observed among individuals vaccinated in November 2024 (46.1%; 95% CI: 36.4-54.6%). <b>Conclusions</b>: Influenza vaccination provided measurable protection during the 2024-2025 season in Wuhan. Greater protection was observed among individuals vaccinated in consecutive seasons or during the current season compared with those vaccinated only in the prior season. Vaccination administered in November was associated with the highest effectiveness, highlighting the importance of appropriate vaccination timing.