A peptide-ferriporphyrin conjugate (Gi-F-CAA) was developed to enhance tumor-specific ferroptosis therapy by leveraging in vivo self-assembly. This conjugate consists of a GPX4 inhibitory peptide, a pH-sensitive linker, and ferriporphyrin, which self-assembles under acidic tumor conditions to improve tumor penetration, endocytosis, and GPX4 inhibition. The assembly-enhanced binding (AEB) effect significantly enhances GPX4 inhibition, increasing oxidative stress from the Fenton reaction and inducing ferroptosis. The conjugate demonstrated strong antitumor activity in multiple models, including bladder cancer, multidrug-resistant breast cancer, and large renal cell carcinoma. It also showed potential for delivering various functional agents. The study highlights the importance of in vivo self-assembly in improving therapeutic efficacy and overcoming chemoresistance. The conjugate exhibited excellent biocompatibility and safety, with no significant toxicity observed in major organs. The findings suggest that this strategy could provide an effective alternative for tumor therapy by inducing ferroptosis.A peptide-ferriporphyrin conjugate (Gi-F-CAA) was developed to enhance tumor-specific ferroptosis therapy by leveraging in vivo self-assembly. This conjugate consists of a GPX4 inhibitory peptide, a pH-sensitive linker, and ferriporphyrin, which self-assembles under acidic tumor conditions to improve tumor penetration, endocytosis, and GPX4 inhibition. The assembly-enhanced binding (AEB) effect significantly enhances GPX4 inhibition, increasing oxidative stress from the Fenton reaction and inducing ferroptosis. The conjugate demonstrated strong antitumor activity in multiple models, including bladder cancer, multidrug-resistant breast cancer, and large renal cell carcinoma. It also showed potential for delivering various functional agents. The study highlights the importance of in vivo self-assembly in improving therapeutic efficacy and overcoming chemoresistance. The conjugate exhibited excellent biocompatibility and safety, with no significant toxicity observed in major organs. The findings suggest that this strategy could provide an effective alternative for tumor therapy by inducing ferroptosis.