In this study, a new eco-friendly carbon nanohybrids was developed by hybridizing carboxymethyl cellulose (CMC) biopolymer with various amounts of multi-walled carbon nanotubes (MWCNTs: 0.2, 0.5, 1, 3, and 5%) to produce hydrophobic CMC–C18@MWCNTs, which was then used as a sorbent material for fat content in fatty food samples analysis. The hydrophobic biopolymers were synthesized using an ultrasound-assisted esterification process, and the physicochemical properties were analyzed using FT-IR, XRD, TGA, FE-SEM, and TEM. The performance of the hydrophobic nanocomposites was evaluated by assessing their ability to remove fat content during polycyclic aromatic hydrocarbon (PAHs) analysis in tuna samples. The results showed that CMC–C18–CNT0.2% provided the best peak shapes and highest recoveries for PAHs compounds, ranging between 74.3 and 89.7%, while CMC–C18–CNT5% had the lowest recoveries, ranging between 0 and 35%. Therefore, the lowest amount of MWCNTs was found to be the most efficient for removing fat content with providing high PAHs recovery, while increasing the MWCNTs percentage increased the hydrophobicity and removed PAHs analytes along with fat content. After the investigation, the method was validated using CMC–C18–CNT0.2% in three various levels: 2, 5 and 10 µg/kg. The obtained results were satisfactory; the average recoveries for all PAHs compounds ranged between 74.3 to 89.7%, and the intra-day precision were estimated by coefficient of variation (%CV), where were less than 10% for all PAHs. The LOD and LOQ were lies between 0.33 to 0.89 µg/kg and 1.12 to 1.92 µg/kg respectively. For the calibration curve linearity, the correlation coefficient (r2) were higher than 0.999 for all PAHs. Overall, the hydrophobic CMC–C18@MWCNTs are a promising, modifiable, and useful material for fatty food analysis.