Simultaneous targeted cancer imaging, therapy and real-time therapeutic monitoring can prevent over- or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near-infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH-activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer-specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real-time imaging. Upon NIR irradiation, R16FP-mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring. All in one: A lysosome-aimed multifunctional nanomicelle (Apt-TNP) was developed by integrating a target-cell-specific aptamer, a pH-activatable fluorescent probe, and a near-infrared photosensitizer. Apt-TNP enables simultaneous cancer imaging, photodynamic therapy, and real-time self-feedback of therapeutic efficacy. ? 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.