The purification of recombinant L-asparaginase II from an Escherichia coli fermentation broth was carried out by using solvent-freeze-out technology on the lab scale. Preliminary crystallization conditions were determined by using batch crystallization screening. The purification of L-asparaginase II was performed afterwards. Protein crystals with rhombic shape were obtained from the solvent-freeze-out crystallization using ethanol. The purity of the crystals was evaluated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) together with UV spectrophotometry. It was found that DNA could be mostly removed by using solvent-freeze-out crystallization. Furthermore, the enzymatic activity was preserved.