To investigate the inhibitory effect of Glycyrrhiza uralensis (G. uralensis) and its monomeric compounds on Nav1.4 voltage-gated sodium channels (VGSCs) and analyze the relationship between the content of its marker compounds and the inhibitory rate. Based on this study, we found that 4 mg/ml ethanol extract of G. uralensis at 30%, 50%, 70% and 90% (v/v) exhibited 77.00 ± 0.03%, 34.75 ± 0.09%, 100.00 ± 0.01% and 2.00 ± 0.01% inhibitory rates on INav1.4 respectively, and 8 mg/ml ethanol extract of G. uralensis at 30%, 50%, 70% and 90% (v/v) exhibited 99.00 ± 0.01%, 97.10 ± 0.02%, 100.00 ± 0.01% and 17.00 ± 0.04% inhibitory rates on INav1.4 respectively. Isoliquiritigenin, echinatin, liquiritin and glycyrrhizic acid exhibited higher inhibitory rates of 39.98 ± 4.55%, 33.20 ± 1.61%, 22.62 ± 0.30% and 20.54 ± 4.82% respectively. However, liquiritigenin, formononetin, neoisoliquiritin and glycyrrhetinic acid exhibited lower inhibitory rates of less than 20%. Further, liquiritin apioside, isoliquiritin and neoliquiritin exhibited almost no effect on INav1.4. These findings showed that glycyrrhizic acid reached a maximum concentration of 49.15 μg/ml, while echinatin had the lowest concentration. The ethanol extract of G. uralensis has significant inhibitory effects on Nav1.4 VGSCs. This may be an important mechanism in the treatment of gastrocnemius spasm and could guide further research regarding material basis and mechanism of the treatment of gastrocnemius spasm with peony and licorice decoction.