Three phase partitioning or TPP consists of proteins’ precipitation due to simultaneous presence of ammonium sulfate and t-butanol. The technique has been successfully used for the partial purification of ficin (EC 3.4.22.3) from Ficus carica latex. In this study, ficin of Mediterranean F. carica was for the first time partially purified and recovered in a single step using TPP. Optimal purification parameters of the system were 40% ammonium sulfate saturation (w/v) with 1.0:0.75 (v/v) ratio of crude extract: t-butanol at pH 7.0, which gave 6.04 purification fold with 167% recovery of ficin. The enzyme was found to be exclusively partitioned in the interfacial precipitate phase. The Tricine SDS–polyacrylamide gel electrophoresis analysis of the enzyme showed comparatively purification and protein molecular weight of the enzyme was nearly found to be 23.4 kDa. Characterization of enzyme showed that optimal pH of purified enzyme was 6.5 and optimal temperature was 60 °C. The enzyme was found to be stable at 40–70 °C for 1 h. This study showed that TPP is a simple, economical and quick method for ficin recovering from F. carica latex trees and can be performed in a purification process to be used successfully in food industries.