The currently available immune checkpoint inhibitors (ICIs) often fail to achieve the desired clinical outcomes due to inadequate immune activation, particularly in patients with lung cancer. To reverse this situation, we synthesized inhalable PFCE-C25 nanoemulsions (NEs), which target lymphocyte activation genes (LAG-3) on immune cells within tumor microenvironment and tumor-draining lymph nodes (TDLNs). By combining in vivo 19F-MR molecular imaging, we investigate the immunological effects of a single low-dose PFCE-C25 NEs in multiple murine lung cancer models, including human immune system (HIS) mouse models, and validated its immunological effects in human TDLNs. The nebulization therapy with PFCE-C25 NEs demonstrated a notable and enduring maturation of dendritic cells (DCs) in TDLNs, leading to systemic immune responses, prolonged survival, the establishment of immune memory, and resistance to tumor rechallenge. Thus, PFCE-C25 NEs successfully demonstrate a promising and efficient approach for enhancing lymphatic transport and sustained activation of antitumor immune responses in lung cancer.