Duckweed contains mainly starch and protein, and protein is a coproduct of starch-based biofuel production. Plant proteins have a great potential for value-added products. However, the lack of information on duckweed protein as a bioenergy coproduct limits its use. In this study, duckweed protein was extracted, purified, and characterized for chemical composition, molecular weight, surface hydrophobicity, emulsion, thermal stability, and rheological properties. Duckweed (Spirodela polyrhiza) with an initial protein content of 34.5% was selected for this study, and the protein was extracted from fresh, frozen, and ambient temperature dried duckweed. The highest extraction rate (52.1%) was obtained from fresh duckweed, followed by ambient-dried duckweed (45.6%) and frozen duckweed (44.3%). The protein samples extracted from ambient-dried duckweed had the highest purity (67.8%) of the three prepared duckweed sources. Duckweed protein showed a low stability of the emulsion and poor emulsifying properties. Molecular weights of duckweed protein fractions ranged from 14 kDa to more than 160 kDa. FTIR showed five distinct absorption bands related to amide A and B, amide I and II, and the carbohydrate component. The melting peak of the duckweed protein sample was broad; it began below 50 degrees C, covered a range of about 120 degrees C, ended at 160 degrees C, and degraded at 250 degrees C. Duckweed protein is more hydrophobic than soy proteins at the same pH value, which suggests that duckweed protein has potential to improve water resistance of protein-based adhesives.