There has been conflicting evidence from meta-analyses on the effect of polyunsaturated fatty acids (PUFA) on non-alcoholic fatty liver disease (NAFLD). Therefore, in this umbrella meta-analysis, we are evaluating whether omega-3 PUFA supplementation has any benefit in treating NAFLD. Electronic databases such as PubMed, Web of Science, Scopus, Embase and Google Scholar were assessed to October 2022. This meta-analysis included all meta-analyses that examined the effect of PUFAs on liver fat and liver function tests [aspartate aminotransferase (AST), alanine aminotransferase (ALT), and Gamma-glutamyl transferase (GGT). Meta-analysis was conducted using a random-effects model. Subgroup analyses and sensitivity analyses were also performed. In total, eight articles involving 6,561 participants met the eligibility criteria. Advantageous impacts PUFA supplementation were observed on ALT (ESWMD = -6.72 IU/L; 95% CI: -8.61, -4.84; p < 0.001, and ESSMD = -0.52 IU/L; 95% CI: -0.84, -0.20, p < 0.001), AST (ESWMD = -3.73 IU/L, 95%CI: -5.93, -1.53, p<0.001, and ESSMD = -0.65 IU/L; 95% CI: -1.08, -0.22, p= 0.003), GGT levels (ESWMD = -4.20 IU/L, 95% CI: -6.85, -1.55, p=0.002), and liver fat (ESWMD = -5.16; 95% CI: -8.49, -1.82, p< 0.001). Intervention with omega-3 PUFAs improves ALT, AST, GGT, and liver fat in patients with NAFLD. Thus, omega-3 PUFAs could be considered as a therapeutic option in the treatment of NAFLD. This article is protected by copyright. All rights reserved.