Platycodin D (PD) has been used as the quality control marker of Radix Platycodonis for its high content and various pharmacological properties. In this study, a specific polyclonal antibody against PD (PD-pAb) was developed, and PD-pAb-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for the detection of PD in Radix Platycodonis. The 50% inhibition concentration (IC50) of PD was 2.70 μg/mL and the linearity range for PD was from 0.032 μg/mL to 100 μg/mL. No cross reactivity with PD-pAb was found in five PD analogs except for platycodin D2 (PD2, 0.93%). The average recovery of PD by icELISA was 97.14% (RSD = 1.17%). The icELISA was used for the detection of PD in different Radix Platycodonis samples and the results were confirmed by high performance liquid chromatography (HPLC). The correlation coefficient between the two assays was 0.9654. Taken together, the established icELISA might be a simple, cheap, rapid, sensitive, reliable and high-throughput method for determining the contents of PD in Radix Platycodonis.