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Protein isolate of sea cucumber guts (SCGPI) was extracted from defatted power of sea cucumber guts (SCGs) by alkaline extraction and acid precipitation method. The physicochemical and functional properties of SCGPI were evaluated in comparison with those of SCGs. It was found that the SCGPI showed higher whiteness, and contained 39.5% of the essential amino acids with higher protein efficiency ratio. The functionalities of SCGPI, including solubility, water/oil holding capacity (WHC/OHC), foaming capacity (FC), and emulsifying abilities (EAI), were significantly higher than those of SCGs. SCGPI exhibited higher total sulfhydryl group, disulfide bond, β‐turns, and random coil, while less surface hydrophobicity and α‐helix than SCGs. The thermal denaturation temperature of SCGPI was 46.55°C and the microstructure of SCGPI was flaky. Moreover, OHC, FC, and EAI of SCGPI were significantly better than those of SPI. These results suggest SCGPI can be used as functional bases in the food industry. Practical applications: Sea cucumber (Stichopus japonicus) guts are the main byproducts in the sea cucumber processing, which are rich in protein. In this study, we prepared protein isolate from sea cucumber guts (SCGPI) by alkaline extraction and acid precipitation method. The results showed that SCGPI exhibited great solubility, water/oil holding capacity, foaming capacity, and emulsifying abilities. The present study demonstrates that SCGPI can be used as a functional base in the food industry. [ABSTRACT FROM AUTHOR]