Overexpressing human NAD(P)H:quinone oxidoreductase 1 (hNQO1) in lung cancer tissues is deemed to be an attractive biomarker, which is directly connected to cancerous pathological processes. Monitoring of hNQO1 activity is crucial to early diagnosis and prognosis of lung cancer. In this study, an activatable hemi-cyanine dye-based probe (denoted as the LET-10 probe) was synthesized for near-infrared fluorescence (NIRF) and ratiometric photoacoustic (RPA) imaging of hNQO1. LET-10 can realize the NIRF and PA signal opening in the presence of hNQO1. Taking the octabutoxy naphthalocyanine in the LET-10 probe as a built-in reference signal, the LET-10 probe further demonstrated a double-signal self-calibration process for RPA imaging. Finally, the LET-10 probe was successfully applied for NIRF/RPA duplex imaging in the hNQO1-positive A549 lung cancer model, which suggests that the LET-10 probe is a promising tool for in vivo hNQO1 detection, especially for lung cancer diagnosis.