Nanotechnology has been widely applied in tumor imaging, diagnostic and therapy. Beside the prefabricated nanomaterials, constructing nanostructures in living cells through self-assemble provides an alternative strategy to treat cancer. In vivo self-assembly renders the conversion of compatible small molecules into assembled nanostructures with toxicity, and is expected to outperform the prefabricated nanotechnologies as the small molecules diffuse faster than their assembly form. Attributed to the specific tumor environment such as low pH, high ROS, high enzyme expression and so on, in vivo self-assembly could differentiate cancer cells from normal ones with high selectivity. The in vivo self-assembly based caner therapy has made considerable progress in the last decade with confirmed advantages such as high capacity, minimal drug resistance, high accumulation, enhanced retention and so on. This review summarized the in vivo self-assembly of nanostructures induced by the stimuli like pH, ROS, enzyme, metal ion, localized concentration, biominerization and their utilization in cancer therapy.