Matrine extraction using reverse micelles of a non-ionic trialkyl phosphine oxide (TRPO) surfactant was studied. Theoretical analysis and experimental results showed that the driving forces for the extraction are the coordination forces between matrines and TRPOs. Oxymatrine and oxysophocarpine cannot form coordination bonds with TRPO, and therefore, they can be separated with matrine from a raw matrine solution. The effect of several important factors such as pH, W-0, and co-solvent on the partition coefficient of matrine was studied. The presence of ions in the system did not affect the partition coefficient significantly. Under the optimum operating conditions, determined by orthogonal experiments, the yield of matrine from the raw matrine solution can reach 70%; the purity of the matrine is higher than 90%. The mass transfer coefficient for both forward and back extraction was calculated and compared with that for reverse micellar extraction of proteins reported in the literature. This paper ends with a discussion of the kinetics of the extraction.