Update information. Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to fight against the widespread SARS-CoV-2 virus, as this coronavirus must bind to ACE2 to allow for its entry into host cells. The huge amount of charge isoforms and complicated charge heterogeneity have posed a challenge to charge variant investigation in pharmaceutical development. To address this, we employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed analysis of enriched ACE2Fc fractions using electrophoretic, chromatographic, mass spectrometric, and biolayer interferometric techniques, for unveiling the structural origins of ACE2Fc charge heterogeneity. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, utilizing the highest allowable voltage to maximize the FF-IEF separation window and pI resolution. Additionally, we preserved the structural integrity of ACE2Fc fractions by implementing a mild elution method for fraction recovery. Based on peptide mapping and other characterizations, we revealed that highly sialylated multi-antenna N-glycosylation significantly contributes to the complex profiles of ACE2Fc charge heterogeneity. In addition, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards SARS-CoV-2 S protein based on the correlation between modifications and the observed activity shift. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a clear sialylation-activity relationship in ACE2Fc enabled by FF-IEF fractionation.