Cysteine (Cys), one of cellular biothiols in the organism, is associated with many diseases, such as Parkinson’s disease, Alzheimer’s disease, liver damage, rheumatoid arthritis, and cancer. However, activatable fluorescence/photoacoustic (FL/PA) probes for non-invasive, real-time, and deep imaging of Cys in vivo are still lacking, and this hinders the diagnosis of Cys-related diseases. Herein, we designed and synthesized a novel activated FL/PA dual-modality cyanine-base probe (FP700) for real-time detection of Cys. The probe FP700 was established with near-infrared emissive dye cyanin as the fluorophore, linking with 2, 4-dinitrobenzenesulfonyl group as the recognition moiety for Cys. Using the FP700, we found that the FP700 exhibited intensive “turn-on” FL/PA signals under the excitation of 700 nm, which realized noninvasive in vivo detection of exogenous Cys. Significantly, FP700 accurately detected endogenous Cys though the FL/PA dual-mode imaging technology in tumor-bearing mice and obtain 3D PA diagnostic images with deep penetration depth and spatial resolution. Thus, the new dual-modality probe FP700 has advantage of high potential for deep tumor diagnosis of Cys in vivo, which may provide a new approach for the detection of cervical cancer and identification of its potential therapeutic targets.