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Nsp13, a non-structural protein belonging to the coronavirus family 1B (SF1B) helicase, exhibits 5′–3′ polarity-dependent DNA or RNA unwinding using NTPs. Crucially, it serves as a key component of the viral replication–transcription complex (RTC), playing an indispensable role in the coronavirus life cycle and thereby making it a promising target for broad-spectrum antiviral therapies. The imidazole scaffold, known for its antiviral potential, has been proposed as a potential scaffold. In this study, a fluorescence-based assay was designed by labeling dsDNA substrates with a commercial fluorophore and monitoring signal changes upon Nsp13 helicase activity. Optimization and high-throughput screening validated the feasibility of this approach. In accordance with the structural characteristics of ADP, we employed a structural-based design strategy to synthesize three classes of imidazole-based compounds through substitution reaction. Through in vitro activity research, pharmacokinetic parameter analysis, and molecular docking simulation, we identified compounds A16 (IC₅₀ = 1.25 μM) and B3 (IC₅₀ = 0.98 μM) as potential lead antiviral compounds for further targeted drug research.