Airborne endotoxin, a bioaerosol component of Gram-negative bacterial cell walls, is a considerable risk to human health. In this study, a systematic optimization and the analysis of corresponding influence mechanism based on the Limulus amebocyte lysate assay were operated by changing sampling duration, sonication pretreatment, extraction solution, and impinger types. Moreover, the corresponding influential mechanisms of these four factors were identified. Experimental results showed that endotoxin concentration tended to increase initially and then declined over time, and that the extraction solution reached saturation after 15 min of sampling. The majority of the cells were disrupted by an ultrasonication pretreatment of less than 800 W, allowing the detection of free endotoxins by the Limulus amebocyte lysate assay. However, a sonication power greater than 800 W could destroy endotoxin structure. Furthermore, the lipophilic and hydrophilic groups in the molecular structure of Tween 20 promoted endotoxin dissolution. Three samplers with different pore sizes and aperture numbers were compared. The results showed that collection efficiency was directly proportional to nozzle aperture size. Small pore sizes and high aperture numbers enhanced airborne endotoxin absorption because they could generating more bubbles with small specific surface area, thereby increasing the interaction between the endotoxins and extraction solution and improving absorption efficiency. Therefore, an optimized sampling method was proposed that collecting air with an AGI-30 impinger and pyrogen-free, sterile purified water (PFW) containing 0.05% Tween 20 at a sampling duration of 10 min. The sample was then sonicated at 800 W for 10 min.