Endoplasmic reticulum (ER) is the largest organelle in eukaryotic cells and plays a variety of functions in living cells include protein folding, calcium homeostasis, and lipid biosynthesis. Normal function of ER is crucial for cell survival, while disequilibrium of ER can cause misfolding of proteins and ER stress, leading to many serious diseases. It has been documented that ER stress is closely related to the metabolism of Cu2+, as ER is the main intracellular accumulation space of Cu2+ and toxic reactive oxygen species can be generated by Cu2+ via Fenton and Haber-Weiss reactions. In this context, developing a powerful tool capable of selective and sensitive monitoring of Cu2+ in ER and investigating its role in physiological and pathological processes is of great importance. Herein, we report the first ER targeted near infrared (NIR) nanosensor, polymer dots encapsulated with NIR hydrophobic carbon nanodots, for detecting Cu2+ in biosystems. This nanosensor with stable fluorescence showed a fast response toward Cu2+ (120 s) and can be used for the quantification of Cu2+ in a linear range covering from 0.25 to 9.0 μM with a detection limit of 13 nM. In addition, the fluorescence variations of the nanosensor are remarkably specific to Cu2+ in comparison with the other metal ions and amino acids. Moreover, the developed nanosensor exhibited low cytotoxicity, good biocompatibility, and ER targeting ability. Because of these excellent spectroscopic features, the nanosensor was successfully utilized for visualizing Cu2+ fluctuations at the living cell, zebrafish and mouse levels, which further proved its potential application in biological systems.