Abstract: Tumor is not a solo performance that only derived from tumor cells, but is rather a pathological imbalance between transformed cells and their surrounding microenvironment. The components in the tumor microenvironment include the blood and lymphatic vascular networks, stromal fibroblasts, infiltrating immune cells, extracellular matrix (ECM), and abundant soluble factors, which play a critical role in supporting tumor malignant progression. Currently, the diagnostic and therapeutic strategies mainly focus on tumor cells, but specifically targeting and regulating the tumor microenvironment have shown a great potential for precise tumor therapy, adding to the therapeutic and diagnostic efficacy. Nanotechnology-based drug delivery systems have the potential to dramatically improve efficacies of tumor therapy and diagnosis, meanwhile reduce the systemic toxicity associated with the current approaches. Therefore, upon deeply exploiting the tumor microenvironment, nanoformulation has emerged as a promising integrated platform that offers great opportunity for targeting tumor cells and their microenvironment and regulating the whole tumor tissue. Rapid progress in the field of molecular self-assembly using various biological molecules has resulted in the smart nanoformulations. Peptides and peptide derivatives, owing to their biocompatibility, sequence designability, easy modification, and biological functionality, have been widely used as building blocks to construct multifunctional drug delivery systems. Recent experiments have suggested various nanoformulations based on modularized construction of peptides with the aim to specifically target and regulate the tumor microenvironment, which will be the emphasis of this review. We also provide our perspectives on the development of combined drug delivery strategies using nanoformulations with controlled surface and interface properties which show considerably promising in the clinic.