Traditional targeting approach utilizing biological ligands has to face the problems of limited receptors and tumor heterogeneity. Herein, a two-step tumor-targeting and therapy strategy based on inverse electron-demand [4+2] Diels-Alder cycloaddition (iEDDA) is described. Owing to the unique acidic tumor microenvironment, an intravenous injection of tetrazine modified pH (low) insertion peptide could efficiently target and incorporate onto various cell surfaces in tumor tissue, such as cancer cells, vascular endothelial cells, and tumor-associated fibroblasts. The receptor-like tetrazine groups with a large amount and homogeneous intratumoral distribution could then serve as the baits to greatly amplify the tumor-targeting ability of indocyanine green (ICG)-loaded and trans-cyclooctene (TCO)-conjugated human serum albumin (HSA) nanoparticles (TCO-HSA-ICG NPs) via iEDDA after the second intravenous injection. Compared with the passive enhanced permeability and retention (EPR) effect and traditional active targeting approaches, the targeting performance and photothermal therapeutic effect based on the two-step strategy are significantly enhanced, while no notable toxicity is observed. As acidity is a characteristic of solid tumor, the two-step strategy can serve as a universal and promising modality for safe and high-performance nanoparticle-based antitumor therapy.