New classes of enzymatic-biodegradable amphiphilic poly(ester-urethane)s were designed and developed from l-tyrosine amino acid resources and their self-assembled nanoparticles were employed as multiple drug delivery vehicles in cancer therapy. The amine and carboxylic acid functional groups in l-tyrosine were converted into dual functional ester-urethane monomers and they were subjected to solvent free melt polycondensation with hydrophilic polyethylene glycols to produce comb-type poly(ester-urethane)s. The phenolic unit in the l-tyrosine was anchored with hydrophobic alkyl side chain to bring appropriate amphiphilicity in the polymer geometry to self-assemble them as stable nanoscaffolds in aqueous medium. The topology of the polymer was found to play a major role on the glass transition, crystallinity, and viscoelastic rheological properties of l-tyrosine poly(ester-urethane)s. The amphiphilic polymers were self-assembled as 200 ± 10 nm nanoparticles and they exhibited excellent encapsulation capabilities for anticancer drugs such as doxorubicin (DOX) and camptothecin (CPT). In vitro drug release studies revealed that the drug-loaded l-tyrosine nanoparticles were stable at extracellular conditions and they underwent enzymatic-biodegradation exclusively at the intracellular level to release the drugs. Cytotoxicity studies in the cervical cancer (HeLa) and normal WT-MEFs cell lines revealed that the nascent l-tyrosine nanoparticles were nontoxic, whereas the CPT and DOX drug-loaded polymer nanoparticles exhibited excellent cell killing in cancer cells. Confocal microscopic imaging confirmed the cellular internalization of drug-loaded nanoparticles. The drugs were taken up by the cells much higher quantity while delivering them from l-tyrosine nanoparticle platform compared to their free state. Flow cytometry analysis showed that the DOX-loaded polymer nanoscaffolds internalized the drugs 8-10× higher compared to free DOX. Both the synthesis of new classes of poly(ester-urethane)s via melt polycondensation approach and the enzyme-responsive drug delivery concept were accomplished for the first time. Thus, the present investigation is expected to open up new opportunities for l-tyrosine polymeric materials in biomaterial and thermoplastic applications.