There is an increasing demand for effective noninvasive diagnosis against common pulmonary diseases, which are rising sharply due to the serious air pollution. Human neutrophil elastase (HNE), a typical protease highly involved in pulmonary inflammatory diseases and lung cancer, is a potential predictor for disease progression. Currently, few of the HNE-targeting probes are applicable in vivo due to the limitation in sensitivity and biocompatibility. Herein, we reported the achievement of in vitro detection and in vivo imaging of HNE by incorporating the HNE-specific peptide substrate, quantum dots (QDs), and organic dyes into the fluorescence resonance energy transfer (FRET) system. The refined nanoprobe, termed QDP, could specifically measure the HNE with excellent sensitivity of 7.15 pM in aqueous solution and successfully image the endogenous and exogenous HNE in living cells. In addition, this nanoprobe enabled HNE imaging in mouse models of lung cancer and acute lung injury, and the HNE activity at high temporal and spatial resolution was continuously monitored. Most importantly, QDP successfully discriminated the serums of patients with lung diseases from those of the healthy controls based on the HNE activity determination. Overall, this study demonstrates the advantages of a FRET-system-based nanoprobe in imaging performance and provides an applicable tool for in vivo HNE detection and pulmonary disease diagnosis.