In urban built-up areas, the water seepage and leakage accidents of karst tunnel occur frequently. However, research on the causal mechanism, disaster-causing mechanism and targeted prevention and control measures is still relatively scarce. Taking the water seepage accident of Chongqing Tuzhu Tunnel as a typical case, the main seepage mechanisms of this tunnel are discussed. Due to the unsmooth surface drainage system, a large amount of surface water accumulates in the collapse pit and seeps down rapidly through the vertical channel, causing a sharp increase in the water pressure outside the tunnel. The fissures are well developed in the surrounding rock of the karst area and there are multiple cavities. Groundwater converges to the tunnel vault through karst channels. The high external water pressure penetrates the secondary lining. The thickness of the water-resistant lining in the tunnel design is insufficient, which makes the drainage channels prone to blockage, and the external water pressure is likely to far exceed the design value, which causes damage to the secondary lining. Based on this, the core ideas for prevention and control measures are proposed to fully consider the risk of karst channels and accurately locate the leakage points and karst channels by using ground-penetrating radar and other technologies. The construction schemes are optimized to reduce the construction disturbance. According to the actual geological conditions, the blocking and drainage mode should be reasonably selected, and the sufficient drainage capacity should be reserved or the strength of the water-resistant lining should be strengthened. The construction quality should be strictly controlled to ensure the effective operation of the waterproofing and drainage system. The comprehensive monitoring and early warning system is established. The prevention measures can provide the beneficial reference for the similar projects, and help improve the construction quality and operational safety of urban karst tunnels.