The stimuli-responsive nanomaterials are gaining more and more interest in the biological field,including cell imaging and biosensing etc. Nanomaterials in response to the bio-relevant stimuli(i.e., p H, enzymes and other bioactive molecules) can be utilized to enhance imaging(i.e., optical imaging, MRI, and multi-mode imaging) sensitivity via disease site-specific delivery and controlled release, which helps to diagnose cancer at an early stage or to monitor progression during treatment. In the triggered responsive process, smart nanomaterials undergo changes in physiochemical properties that can cause cytotoxicity or influence on cell functions due to the interactions between nanomaterials and cells. In order to promote the design and fabrication of effective platforms for therapeutics and diagnostics, special attention should be paid to these effects. By taking the advantages of intracellular stimuli, the controlled self-assembly in living cells can be achieved, which has been used for various in situ detections and insights into biological self-assembly. In this review, the recent advances in cell imaging, cytotoxicity and self-assembly of intracellular stimuli-responsive nanomaterials are summarized. Some principles for the further design and applications of intracellular stimuli-responsive nanomaterials and future perspectives are discussed. The stimuli-responsive nanomaterials are gaining more and more interest in the biological field, including cell imaging and biosensing etc. Nanomaterials in response to the bio-relevant stimuli (ie, p H, enzymes and other bioactive molecules) can be utilized to enhance imaging (ie, optical imaging, MRI, and multi-mode imaging) sensitivity via disease site-specific delivery and controlled release, which helps to diagnose cancer at an early stage or to monitor progression during treatment. In the triggered responsive process, smart nanomaterials undergo changes in physiochemical properties that can cause cytotoxicity or influence on cell functions due to the interactions between nanomaterials and cells. In order to promote the design and fabrication of effective platforms for therapeutics and diagnostics, special attention should be paid to these effects. By taking the advantages of intracellular stimuli, the controlled self-assembly in living cells can be achieved, which has been used for various in situ detections and insights into biological self-assembly. In this review, the recent advances in cell imaging, cytotoxicity and self-assembly of intracellular stimuli-responsive nanomaterials are summarized. Some principles for the further design and applications of intracellular stimuli- responsive nanomaterials and future perspectives are discussed.