Active-targeting nanomedicines have been widely employed in cancer treatment for increasing therapeutic index. However, the limited permeability caused by the binding site barrier (BSB) and size hindrances compromises their clinical antitumor efficacy in patients. Herein, learning from the liquid-liquid phase separation (LLPS) of bio-macromolecules, we report phase-separating glycopeptides (HEP) from polyhistidine (PHis) grafted hyaluronic acid (HA), which can sense the tumor extracellular pH and concomitantly overcome size and BSB dilemmas for enhanced tumor penetration. HEP aggregates into nanodroplets in solution at neutral pH. Upon reaching the acidic extracellular environment of tumors, the pH-responsive PHis triggers a phase separation, converting the coacervate nanodroplets into monomeric glycopeptides. This enables HEP conjugated with the platinum prodrug (HEPPt) to deeply penetrate into tumors by overcoming the BSB effect arising from the interaction between nanodroplets and cluster of differentiation 44 (CD44), as well as resolving the size challenges. Moreover, HEPPt in monomeric states exhibits promoted cellular uptake after pH-triggered phase separation, attributed to the transmembrane effect of exposed PHis. Subsequently, the rapid release of Pt(II), triggered by tumor intracellular reducing environment, exerts excellent antitumor activity. The phase-separating glycopeptides represent a promising platform for improving tumor penetration and intracellular delivery of therapeutic agents.