Sonoporation has been shown to have an important role in biotechnology for gene therapy and drug delivery. This paper presents a novel microfluidic sonoporation system that achieves high rates of cell transfection and cell viability by operating the sonoporation chamber at resonance. The paper presents a theoretical analysis of the resonant sonoporation chamber design, which achieves sonoporation by forming an ultrasonic standing wave across the chamber. A piezoelectric transducer (PZT 26) is used to generate the ultrasound and the different material thicknesses have been identified to give a chamber resonance at 980 kHz. The efficiency of the sonoporation system was determined experimentally under a range of sonoporation conditions and different exposures time (5, 10, 15, and 20 s, respectively) using HeLa cells and plasmid (peGFP-N1). The experimental results achieve a cell transfection efficiency of 68.9% (analysis of variance, ANOVA, p < 0.05) at the resonant frequency of 980 kHz at 100 V(p-p) (19.5 MPa) with a cell viability of 77% after 10 s of insonication.