Abstract: This study investigated the effects of different ethanol concentrations (ranging from 0% to 60%) on the structural and physicochemical properties of sea cucumber collagen, aiming to elucidate the ethanol–collagen interaction mechanism and provide a theoretical basis for quality control in ethanol-containing food systems. To achieve a comprehensive understanding of these effects, we systematically measured the solubility, particle size, total sulfhydryl content, and dityrosine content of the collagen samples treated with different ethanol concentrations. Furthermore, the conformational changes of the collagen were analyzed by combining multidimensional fluorescence spectroscopy (intrinsic, synchronous, and three-dimensional fluorescence) with Raman spectroscopy. The obtained fluorescence spectra results demonstrated that as the ethanol concentration increased, the collagen solubility exhibited a significant downward trend, decreasing by 39.9% when treated with 60% ethanol compared to the control group. Conversely, the particle size increased significantly, showing a 150.4% increase at 60% ethanol, indicating progressive aggregation. The contents of total sulfhydryl and dityrosine exhibited a biphasic trend, initially increasing and then decreasing, both reaching their peak values when treated with 40% ethanol. The intrinsic fluorescence intensity reached its maximum at 40% ethanol treatment and exhibited a blue shift phenomenon at higher concentrations. Synchronous fluorescence analysis indicated that ethanol primarily affected the microenvironment of tryptophan residues. This change was consistent with conformational rearrangement and further confirmed the specific interaction site of ethanol on collagen. Additionally, secondary structure analysis showed that α-helix content decreased. At 60% ethanol, α-helix content declined by 70.99%. Meanwhile, β-turn content and random coil content increased. These changes demonstrated a loss of ordered helical structure and a gain in less ordered or differently ordered structural elements. The secondary structure transition provided molecular evidence that ethanol altered the folding state of collagen. The results from laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) confirmed that high concentrations of ethanol induced collagen aggregation, leading to the formation of irregular large particles. Meanwhile, the differential scanning calorimetry (DSC) results showed that ethanol affected the thermal stability of collagen in a concentration-dependent manner, first enhancing and then weakening it, with the maximum denaturation temperature (T_max) being highest at 40% ethanol. This trend was consistent with the changes in secondary structure and microstructure. This indicated that low to moderate ethanol treatment strengthened thermal stability, whereas higher ethanol treatment weakened it. In summary, ethanol significantly affects the structural stability of sea cucumber collagen by destroying hydrogen bonds, altering hydrophobic interactions, and inducing secondary structure transitions. Low ethanol concentrations (≤40%) moderately unfold the protein, exposing buried hydrophobic and sulfhydryl groups, which enhanced intermolecular interactions and thermal stability. High concentrations (>40%) exacerbated denaturation and aggregation, causing irreversible structural damage, reduced solubility, and decreased thermal stability. This study provides a theoretical basis for controlling protein behavior in ethanol-containing food systems and practical guidance for optimizing ethanol concentration in products such as wine-flavored sea cucumber, with 40% ethanol identified as a critical threshold for maintaining quality and stability.