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This research reports for the first time on the application of agarose gel impregnated with high fructose corn syrup (AG/HFCS) as a biodegradable and eco-friendly extraction phase in rotating-disk sorptive extraction (RDSE). The gel disk is driven by a rotary rod attached to an electric stirrer during extraction. Malondialdehyde (MDA) was chosen as a model analyte, and was extracted from biological and food samples using the proposed technique after derivatization with 2-thiobarbituric acid (TBA). Due to the hydrophilic nature of the sorbent phase, MDA was concentrated efficiently. After extraction, MDA was quantified directly in the gel disk by solid phase visible spectrophotometry and smartphone-based Red-Green-Blue (RGB) detection. The procedure used no organic solvents, showed clear advantages in terms of simplicity and short analysis time (5 min), and showed potential as a green analytical method. The extraction procedure was studied and optimized to maximize the partition of MDA into the gel. Under optimized conditions, the method provided linear dynamic ranges of 5.5–1000 ng mL−1 for biological samples and 62.5–12500 ng g−1 for food samples with correlation coefficients (R2) higher than 0.9975, relative recoveries between 88.3 and 103.3% along with relative standard deviation (RSD) values less than 3.5%. Accordingly, the proposed method can be employed by analytical laboratories for the rapid determination of MDA in complex matrix of body fluids and food samples under the principles of green chemistry.