The ability to identify cancer lesions with endogenous biomarkers is currently limited to tumours ~1 cm in diameter. We recently reported an exogenously administered tumour-penetrating nanosensor that sheds， in response to tumour-specific proteases， peptide fragments that can then be detected in the urine. Here， we report the optimization， informed by a pharmacokinetic mathematical model， of the surface presentation of the peptide substrates to both enhance on-target protease cleavage and minimize off-target cleavage， and of the functionalization of the nanosensors with tumour-penetrating ligands that engage active trafficking pathways to increase activation in the tumour microenvironment. The resulting nanosensor discriminated sub-5 mm lesions in human epithelial tumours and detected nodules with median diameters smaller than 2 mm in an orthotopic model of ovarian cancer. We also demonstrate enhanced receptor-dependent specificity of signal generation in the urine in an immunocompetent model of colorectal liver metastases， and activation of the nanosensors in human tumour microarrays when re-engineered as fluorogenic zymography probes.