Objective To explore the clinical value of high-frequency ultrasound Hepatosplenic Index （HSI） in the grading diagnosis of non-alcoholic fatty liver （NALFD）. Methods Retrospectively, 233 patients with liver disease from five hospitals nationwide were included, all of whom underwent surgery or liver biopsy to obtain pathological grading of fatty liver. Based on the pathological results, the study classified the subjects into the normal group(27 cases), mild group(77 cases), moderate group(83 cases), and severe group(46 cases).All patients underwent ultrasound examination within 14 days before obtaining the pathological results, and the ratio of the average gray values of the liver and spleen was defined as HSI. Clinical data were collected to compare the differences in the above parameters among the groups. Multiple linear regression analysis was used to analyze the correlation between HSI and clinical data and the degree of fatty degeneration. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic performance of HSI for different degrees of hepatic steatosis. Results The differences in age, AST, ALB, TC, LDL, and TG between the mild, moderate, severe groups, and the normal group were statistically significant (all P < 0.05). The HSI values in the normal, mild, moderate, and severe groups were 1.04±0.09, 1.11±0.14, 1.22±0.13, and 1.33±0.17, respectively, with statistically significant differences among the groups (P < 0.05). The correlation analysis showed that HSI was linearly positively correlated with the degree of fatty degeneration (β=0.115, t=22.824, P＜0.001). ROC curve analysis showed that the area under the curve（AUC） for HSI in diagnosing the presence or absence of fatty liver, moderate-to-severe fatty liver, and severe fatty liver were 0.84（95% CI 0.76-0.91）, 0.86（95% CI 0.81-0.91）, and 0.80（95% CI 0.72-0.88）, respectively；The optimal cutoff points for diagnosing fatty liver, moderate-to-severe fatty liver, and severe fatty liver using HSI were 1..13（Sen：60.2%, Spe：92.3%）, 1.16（Sen：82.0%, Spe：92.4%）, and 1.24（Sen：78.3%, Spe：86.6%）, respectively. Conclusion HSI of high-frequency ultrasound has important clinical value in the grading diagnosis of NAFLD.