Protein-polymer conjugation is a clinically validated approach to enhanced pharmacokinetic properties. However, the permanent attachment of polymers often leads to irreversibly reduced protein bioactivity and poor tissue penetration. As such, the use of protein-polymer conjugates for solid tumors remains elusive. Herein, we report a simple strategy using enzyme-activatable and size-shrinkable protein-polypeptide conjugates to overcome this clinical challenge. Briefly, a matrix metalloproteinase (MMP)-responsive peptide sequence is introduced between a therapeutic protein interferon (IFN) and a synthetic polypeptide P(EG3Glu)20. The resulting site-specific MMP-responsive conjugate, denoted as PEP20-M-IFN, can, therefore, release the attached P(EG3Glu)20 to achieve both protein activation and deep penetration into the tumor microenvironment (TME). Compared to a similarly produced nonresponsive analogue conjugate PEP20-IFN, our results find PEP20-M-IFN to show higher bioactivity in vitro, improved tumor retention, and deeper penetration in a MMP2-dependent manner. Moreover, systemic administration of PEP20-M-IFN shows outstanding antitumor efficacy in both OVCAR3 and SKOV3 ovarian tumor models in mice. This work highlights the releasable PEPylation strategy for protein drug potentiation at the TME and opens up new opportunities in clinics for the treatment of malignant solid tumors.