Abstract: Ultra-high-performance concrete (UHPC) is a cement-based composite material with ultra-high strength, ultra-strong durability, and ultra-high toughness. Its internal mesoscopic defects can accelerate the corrosion of steel fibers and seriously affect the strength of UHPC structures. In this study, based on the ultrasonic transmission method of non-destructive testing, four groups of UHPC test blocks with different porosities and pore sizes, prefabricated using expandable polystyrene (EPS) particles, were experimentally investigated. The influence of mesoscopic pore defects on the amplitude, integral area, and spectral peak value of UHPC acoustic waves was explored, and the experimental results were analyzed and verified through finite element simulation. The results show that as pore size and porosity increase, the maximum amplitude in the time domain of the acoustic wave decreases, the integration area in the time domain reduces, and the spectral peak shifts toward lower frequencies, with a decrease in peak value and the occurrence of multimodal phenomena. The amplitude change can be converted into an applicable acoustic wave parameter, the energy reception ratio En, which can effectively evaluate the presence of mesoscopic defects in UHPC. The change trend of En is opposite to that of porosity and pore size. Finite element simulation confirms the same attenuation law.