The development of lung-specific conditional knockout animal models is pivotal in advancing lung disease research. This protocol describes the experimental procedures in detail, including the construction of loxP mice, endotracheal tube nebulization of adeno-associated virus (AAV) and whole lung clearing. Mouse lung conditional knockouts were successfully constructed. This meticulous approach allows for accurate control over the timing and location of the knockout, facilitating an in-depth investigation into the pathogenesis of lung diseases and the development of therapeutic strategies. These lung-specific conditional knockout models represent a powerful tool for the study of lung diseases, offering valuable insights and guidance for the advancement of treatment approaches and drug discovery in the future.