The single step three-phase partitioning (TPP) method was evaluated for the purification of invertase from Momordica charantia. Optimum conditions for TPP method, that is, 70% ammonium sulfate saturation, 1:0.5 t-butanol ratio and 30 min incubation time lead to 20.28% efficiency and 10.48 fold partial purification. Total protein decreased from 34.86 mg to 0.68 mg in the homogenate, and the specific activity of the enzyme increased from 21.67 to 227.02 U/mg protein. The purified enzyme showed maximum activity in 0.3 M pH 5.0 buffer at 50°C. In conclusion, the optimized single step TPP method revealed better purification.