The activation of retinoic acid-inducible gene I (RIG-I), an indispensable viral RNA sensor in mammals, is subtly regulated by ubiquitination. Although multiple ubiquitination sites at the amino terminus of RIG-I have been identified, their functional allocations in RIG-I activation remain elusive. We identified a stratified model for RIG-I amino-terminal ubiquitination, in which initiation at either Lys164 or Lys172 allows subsequent ubiquitination at other lysines, to trigger and amplify RIG-I activation. Experimental andmathematicalmodeling showed that multisite ubiquitination provides robustness in RIG-I-mediated type I interferon (IFN) signaling. Furthermore, the flexibly controlled ultrasensitivity and IFN activation intensity determine the specificity of the IFN-stimulated gene transcription and manipulate cell fate in antiviral immune response. Our work demonstrates that tunable type I IFN signaling can be regulated through multisite RIG-I ubiquitination and elucidates a new paradigm for dynamic regulation in RIG-I-mediated antiviral signaling.