Biomaterials aim to address healthy issues and contribute to improve life quality for human beings. Currently available biomaterials have been challenged in performing theranostic objectives under real dynamic physiological conditions and at precise targeting sites. To address this concern, over the past few years in situ formulation of biomaterials has been developed to perform disease diagnosis and therapy in a precise manner involving different components. In this review, we introduced the concept of in situ-formed biomaterials and their design principles, specifically summarizing the progress of in situ-formed biomaterials based on stimulusresponsive self-assembly of peptides in living systems. We highlighted the recent examples of in situ assembling systems of peptides with applications ranging from cancer therapy, anti-inflammation and anti-bacteria, as well as tissue engineering and regeneration. The challenges met by in situ biomaterials and the prospects of in situ peptide assembly towards biomedicines are also discussed, which hopefully elucidates the great potential of in situ-formed biomaterials for future healthcare.