The development of a bidentate aptamer-functionalized polydiacetylene (PDA) liposome sensor that is capable of specific colorimetric detection of proteins, directly in complex mixtures (e.g., serum), at sub-micromolar concentrations within 15 min, is reported. In comparison to sensors fabricated with a single aptamer reagent, the conjugation of bidentate aptamer pairs that recognize two distinct exosites of the target protein (thrombin) to the liposome results in significant enhancements of the sensitivity and the specificity. To elucidate the mechanism behind this enhancement, experimental evidence is presented that suggests that the liposomic aggregation triggered by specific, multi-site binding to the target protein is responsible for the improved colorimetric response. Since the colorimetric protein sensor does not require any power or instrumentation, it offers a promising approach towards molecular diagnostics at point-of-care (POC), especially in low-resource settings.