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Enzymatic preparation of low-molecular-weight chondroitin sulfate (LMWCS) has received increasing attention. In this work, a chondroitin sulfate lyase ABC (Chon-ABC) was successfully cloned, expressed, and characterized. The <i>K_m</i> and <i>V_max</i> of the Chon-ABC were 0.54 mM and 541.3 U mg⁻¹, respectively. The maximal activity was assayed as 500.4 U mg⁻¹ at 37 °C in pH 8.0 phosphate buffer saline. The half-lives of the Chon-ABC were 133 d and 127 min at 4 °C and 37 °C, respectively. Enzymatic preparation of LMWCS was performed at room temperature for 30 min. The changes between the substrate and product were analyzed with mass spectrometry (MS), high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR). Overall, the Chon-ABC from <i>Bacteroides thetaiotaomicron</i> is competitive in large-scale enzymatic preparation of LMWCS for its high activity, stability, and substrate specificity.