Abstract: As the global demand for sustainable plant-based food alternatives surges, rapeseed meal has emerged as a promising protein resource owing to its well-balanced amino acid profile. However, its high-value utilization remains severely hindered by the limitations of the conventional alkali-soluble acid precipitation method. This harsh method induces severe protein denaturation and accelerates the oxidation of co-extracted phenolic compounds, ultimately resulting in intense dark browning of the protein products and considerable deterioration in their functional attributes for food applications. To investigate the effects of preparation methods on the structural and functional properties of rapeseed protein, as well as the underlying structure-function relationship, dehulled cold-pressed rapeseed meal was employed as the raw material in this study, and two distinct rapeseed protein products were prepared via the salt-soluble dialysis method and the alkali-soluble acid precipitation method, respectively. Given that preparation processes play a pivotal role in determining protein structural integrity and subsequent processing performance, a systematic characterization and comparative analysis of the multi-scale structural characteristics and functional properties of the obtained rapeseed proteins were conducted using a series of analytical techniques, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared (FTIR) spectroscopy, fluorescence spectroscopy, scanning electron microscopy (SEM), as well as rheological property and processing performance determinations. The results demonstrated that the rapeseed protein prepared via salt-soluble dialysis (SD-RP) held obvious advantages over the alkali-soluble acid precipitation counterpart (AA-RP) in multiple quality indexes. Specifically, SD-RP exhibited more favorable color quality with a lightness value (L* = 44.47), higher protein purity (81.39%), and lower contents of anti-nutritional factors such as polyphenols (34.22 mg/g), that is, the mild conditions of salt-soluble dialysis successfully minimized the co-extraction and oxidation of polyphenols. In terms of protein structure, SD-RP was predominantly composed of native globulin monomers and albumin, retaining a higher proportion of ordered secondary structure (37.86% β-sheet and 20.34% α-helix), in sharp contrast to the severely denatured AA-RP, which only contained 28.06% β-sheet and 14.61% α-helix. Furthermore, intrinsic fluorescence spectroscopy revealed that SD-RP maintained a compact, native tertiary folding conformation with a maximum fluorescence emission wavelength (λ_max) of 325 nm. Conversely, AA-RP suffered from severe molecular unfolding and structural denaturation, with a red-shifted λ_max of 345 nm due to the strong alkali and acid treatment conditions. What’s more, the well-preserved native structure of SD-RP favored the formation of a highly rigid and elastic gel network dominated by a high storage modulus (G’), endowing the protein with exceptional foaming capacity of 185.67%. On the contrary, the unfolded tertiary structure and enhanced molecular chain flexibility of AA-RP conferred superior interfacial properties, enabling faster protein adsorption and rearrangement at the oil-water interface, leading to superior emulsifying activity of 7.73 m²/g and emulsifying stability (84.37%). In conclusion, this study unravels the intrinsic mechanism by which extraction-induced multiscale structural alterations mediate the functional performance of rapeseed protein. And SD-RP is more suitable for food systems requiring high-elasticity gels networks and excellent foaming properties, while AA-RP exhibits greater application potential in interface-dominated food formulations. These findings provide a robust theoretical foundation for the targeted preparation of high-quality rapeseed proteins products to meet diversified, application-specific requirements in the food industry.