A new method—negative pressure cavitation extraction (NPCE) was proposed and investigated for the extraction of the main isoflavonoids, namely genistein and genistin from pigeon pea roots. The effects of extraction time and particle size on the extraction yields were firstly optimized, then a central composite design (CCD) combined with response surface methodology (RSM) was used to study the effects of negative pressure, ethanol concentration and liquid/solid ratio on the extraction yields. The maximum extraction yields of genistein and genistin reached 0.418 and 0.398mg/g, respectively, under the optimal conditions: extraction time 45min, particle size 50 mesh, negative pressure −0.05MPa, ethanol concentration 70% and liquid/solid ratio 44:1. Furthermore, the antioxidant activity of NPCE extract was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay. NPCE extract possessed notable concentration-dependent antioxidant activity with IC 50 value of 0.062mg/ml.