Abstract: 12-N-Benzenesulfonyl-11-matrinic acid derivatives are a new class of anti-CVB3 compounds, but the mechanism of action is still unknown. Therein, two kinds of molecule probes were designed and constructed in this study, including matrinic amines that might be applied to the BIAcore fishing technique and biotin-tagged matrinic derivatives, which could be applied in the biotin affinity chromatography. Moreover, their anti-CVB3 activities were evaluated. Among them, 10a displayed a good activity with an IC₅₀ value of 0.8 μmol·L^-1. This active molecule probe provides a key chemical tool for exploration of the anti-CVB3 mechanism of this type of compounds.