Objectives-Metabolic dysfunction-associated steatotic liver disease (MASLD) isthe most prevalent liver disorder in Western countries, with approximately 20%-30% of the MASLD patients progressing to severe stages. There is an urgentneed for noninvasive, cost-effective, widely accessible, and precise biomarkers toevaluate liver steatosis. This study aims to assess and compare the diagnostic per-formance of a novel reference frequency method-based ultrasound attenuationcoefficient estimation (ACE) in both fundamental (RFM-ACE-FI) and harmonic(RFM-ACE-HI) imaging for detecting and grading liver steatosis. Methods-An Institutional Review Board-approved prospective study was car-ried out between December 2018 and October 2022. A total number of 130 sub-jects were enrolled in the study. The correlation between RFM-ACE-HI valuesand magnetic resonance imaging proton density fat fraction (MRI-PDFF), aswell as between RFM-ACE-FI values and MRI-PDFF were calculated. The diag-nostic performance of RFM-ACE-FI and RFM-ACE-HI was evaluated usingreceiver operating characteristic (ROC) curve analysis, as compared to MRI-PDFF. The reproducibility of RFM-ACE-HI was assessed by interobserveragreement between two sonographers. Results-A strong correlation was observed between RFM-ACE-HI and MRI-PDFF, withR=0.88 (95% confidence interval [CI]: 0.83-0.92;P< .001),while the correlation between RFM-ACE-FI and MRI-PDFF wasR=0.65(95% CI: 0.50-0.76;P< .001). The area under the ROC (AUROC) curve forRFM-ACE-HI in staging liver steatosis grades of S >= 1 and S >= 2 was 0.97 (95%CI: 0.91-0.99;P< .001) and 0.98 (95% CI: 0.93-1.00;P< .001), respectively,and 0.76 (95% CI: 0.65-0.85) and 0.80 (95% CI: 0.70-0.88) for RFM-ACE-FI,respectively. Great reproducibility was achieved for RFM-ACE-HI, with an inter-observer agreement ofR=0.97 (95% CI: 0.94-0.99;P< .001). Conclusions-The novel RFM-ACE-HI method offered high liver steatosis diag-nostic accuracy and reproducibility, which has important clinical implications forearly disease intervention and treatment evaluation