In this study, the effects of chemical refining on the physicochemical indices, environmental pollutants, lipid functional groups, relaxation characteristics, fatty acid profiles, and triglyceride (TG) molecular species in crude oil extracted from small fish in Mauritania were evaluated. The acid, peroxide, and iodine values of crude oil were identified as 5.52 mg KOH/g, 1.73 meq/kg, and 200.78 g/100 g oil, respectively. After refining, these values were determined to be 0.29 mg KOH/g, 0.76 meq/kg, and 210.80 g/100 g oil, respectively. The polychlorinated biphenyls and arsenic content of crude oil were identified as 32.30 μg/kg and 1.00 mg/kg, respectively. After refining, these were not detected. The content of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) accounted for as much as 87.55% of the polyunsaturated fatty acids in the refined oil, indicating that high-quality fish oil is suitable as a food supplement. The correlation coefficient of the infrared spectra before and after refining was 0.968, and the relaxation time and peak shape data were almost similar before and after purification. Meanwhile, there were no significant differences (P ≥ 0.05) in fatty acid profiles between the crude and refined oils, except for C16:0 (P < 0.01). Based on UPLC-Q E-MS/MS detection, 137 TG molecules were identified in both crude and refined oils. Among the detected TG molecular species, 64 contained EPA or DHA, which accounted for 46.83% and 46.00% of the total TG content in crude and refined oils, respectively. After refining, the content of the molecular species decreased by 31.95 mg/10 g, among which the three most variable molecular species were TG (16:0/18:1/22:6), (16:1/16:2/18:1), and (18:4/16:0/20:5), accounting for 29.26% of the decline of the molecular species. Overall, the refining process had little effect on the fatty acid profile and distribution of TG molecular species in fish oil.