The molecular weight of lignin extracted from lignocellulosic biomass is an important factor in determining its valorization in industrial processes. Herein, this work aims to explore the extraction of high molecular weight and bioactive lignin from water chestnut shells under mild conditions. Five kinds of deep eutectic solvents were prepared and applied to isolate lignin from water chestnut shells. The extracted lignin was further characterized with element analysis, gel permeation chromatography, and Ultraviolet-visible and Fourier-transform infrared spectroscopy. The distribution of pyrolysis products was identified and quantified with thermogravimetric analysis—Fourier-transform infrared spectroscopy and pyrolysis-gas chromatograph-mass spectrometry. The results showed that choline chloride/ethylene glycol/p-toluenesulfonic acid (1:1.8:0.2 molar ratio) exhibited the highest fractionation efficiency for lignin (84.17% yield) at 100 °C for 2 h. Simultaneously, the lignin showed high purity (90.4%), high relative molecular weight (37,077 g/mol), and excellent uniformity. Furthermore, the aromatic ring structure of lignin remained intact, consisting mainly ofp-hydroxyphenyl, syringl, and guaiacyl subunits. The lignin generated a large number of volatile organic compounds during the depolymerization process, mainly composed of ketones, phenols, syringols, guaiacols, esters, and aromatic compounds. Finally, the antioxidant activity of the lignin sample was evaluated with the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay; the lignin from water chestnut shells showed excellent antioxidant activity. These findings confirm that lignin from water chestnut shells has a broad application prospect in valuable chemicals, biofuels and bio-functional materials.