The immense potential of carbon nanoprobes (CNPs) for using as contrast agents has propelled much recent research and development in the field of thermoacoustic (TA) molecular imaging, while the proper engineering and design of such materials with required high TA conversion efficiency is still a highly challenging task. In this work, we proposed a controllable strategy to amplify the TA conversion efficiency of the CNPs by constructing vacancy defect (VD) dipoles, and systematically demonstrated the amplification mechanism through theoretical and experimental investigations. First-principles calculation results indicate that, when a carbon atom is removed from the CNPs by chemical approach, owing to local electron density redistribution, the VDs are formed at the positions of the original carbon atoms and act as the structural origin of permanent electric dipoles with the dipole moment several orders higher than that of non-defect sites. Under pulsed microwave irradiation, the VD dipoles are polarized repeatedly and significantly contribute to the conversion efficiency from absorbed electromagnetic waves to ultrasound through enhanced dielectric relaxation losses. We experimentally synthesized graphene samples with different VD densities and VD types to demonstrate the efficiency of the proposed strategy, and results coincide well with the theoretical proposition. This work offers feasible guidance to the systematic development and rational design of new high-conversion-efficiency TA CNPs via VD engineering.