Thermoelectric (TE) performance of Ca₃Co₄O₉ (CCO) has been investigated extensively via a doping strategy in the past decades. However, the doping sites of different sublayers in CCO and their contributions to the TE performance remain unrevealed because of its strong correlated electronic system. In this work, Sr and Ti are chosen to realize doping at the [Ca₂CoO₃] and [CoO₂] sublayers in CCO. It was found that figure of merit (ZT) at 957 K of Ti-doped CCO was improved 30% than that of undoped CCO whereas 1 at% Sr doping brought about a 150% increase in ZT as compared to undoped CCO. The significant increase in electronic conductivity and the Seebeck coefficient are attributed to the enhanced carrier concentration and spin-entropy of Co⁴⁺ originating from the Sr doping effects in [Ca₂CoO₃] sublayer, which are evidenced by the scanning electron microscope (SEM), Raman, Hall, and X-ray photoelectron spectroscopy (XPS) analysis. Furthermore, the reduced thermal conductivity is attributed to the improved phonon scattering from heavier Sr doped Ca site in [Ca₂CoO₃] sublayer. Our findings demonstrate that doping at Ca sites of [Ca₂CoO₃] layer is a feasible pathway to boost TE performance of CCO material through promoting the electronic conductivity and the Seebeck coefficient, and reducing the thermal conductivity simultaneously. This work provides a deep understanding of the current limited ZT enhancement on CCO material and provides an approach to enhance the TE performance of other layered structure materials.