Tropomyosin (TM) and arginine kinase (AK) are known as two major allergens in seafood. For the first time, we demonstrate a newly developed ion-exchange chromatography coupled with dynamic coating capillary electrophoresis (IEC-DCCE) method to simultaneously analyze the TM and AK in shellfish. First, we have optimized the procedure of IEC for simple enrichment of TM and AK crude extract. By using 30 mM borate-borax at pH 9.0 with 0.3% (v/v) Tween-20 as a dynamic coating modifier for capillary electrophoresis (CE) separation, the migration time, separation efficiency and electrophoretic resolution greatly improved. The limits of detection (LOD) were 1.2 µg mL−1 for AK and 1.1 µg mL−1 for TM (S/N = 3), and the limits of quantification (LOQ) were 4.0 µg mL−1 for AK and 3.7 µg mL−1 for TM (S/N = 10). The recovery of AK ranged from 91.5 to 106.1%, while that of TM ranged from 94.0 to 109.5%. We also found that only when the concentrations of AK and TM were above LOD reported here, these proteins can stimulate human mast cell (LAD2) degranulation. Finally, the use of IEC-DCCE to analyze fresh shellfish samples highlights the applicability of this method for the simultaneous detection of these allergens in complex food systems.