Highly efficient isolation and recovery of viable circulating tumor cells (CTCs) from the blood of patients is an important precondition to address the current dilemma of insufficient CTC studies, and can promote the development of individualized antitumor therapies. Herein, a cell-friendly CTC isolation and recovery nanoplatform with luminescent labelling was established using a layer-by-layer (LbL) assembly technique and a stimulated cellular-release strategy. In particular, the anti-epithelial cell adhesion molecule (anti-EpCAM) antibody was introduced with a disulfide bond-containing linker for further bio-friendly recovery of the CTCs. Quantum dots (QDs) were deposited onto fast magnet-responsive Fe3O4 nanoparticles through a facile LbL assembly method to monitor the capture and recovery process in real time. The obtained PEGlyated immuno-magnetic nanospheres (PIMNs) can all be magnetically collected within 2 min. Capture efficiencies above 90% can be achieved from blood samples with 5-200 CTCs per mL after only 1-2 min incubation. Nearly all PIMNs on the surface of the CTCs were detached after 15 min of glutathione (GSH) treatment with the disappearance of QD signals. Recovered CTCs could be directly used for culture (cell viability, ∼98%), and their invasiveness and migration characteristics remained unchanged. Furthermore, the PIMNs were successfully applied to isolate CTCs in cancer patients' peripheral blood samples, and an average of 8.6 ± 5.8 CTCs per mL was detected. The results above suggested that PIMNs may serve as a powerful nanoplatform for CTC screening, isolation and recovery.